U.S. patent application number 11/690627 was filed with the patent office on 2007-12-20 for extracts and methods comprising cinnamon species.
Invention is credited to Randall S. Alberte, Robert T. Gow, Dan Li, George W. Sypert.
Application Number | 20070292540 11/690627 |
Document ID | / |
Family ID | 38523339 |
Filed Date | 2007-12-20 |
United States Patent
Application |
20070292540 |
Kind Code |
A1 |
Gow; Robert T. ; et
al. |
December 20, 2007 |
Extracts and Methods Comprising Cinnamon Species
Abstract
The present invention relates to extracts of cinnamon species
plant material prepared by supercritical CO.sub.2 extractions
methods.
Inventors: |
Gow; Robert T.; (Naples,
FL) ; Li; Dan; (Singapore, SG) ; Sypert;
George W.; (Naples, FL) ; Alberte; Randall S.;
(Estero, FL) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Family ID: |
38523339 |
Appl. No.: |
11/690627 |
Filed: |
March 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60785012 |
Mar 23, 2006 |
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60873475 |
Dec 7, 2006 |
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Current U.S.
Class: |
424/739 ;
426/655; 536/123.1; 536/8; 549/397; 549/403; 549/406; 562/405;
568/425; 568/716; 568/717; 568/840; 585/22 |
Current CPC
Class: |
A61K 36/54 20130101;
C07C 51/47 20130101; A61P 31/10 20180101; A23L 33/105 20160801;
A61P 31/12 20180101; A61P 25/02 20180101; Y02P 20/544 20151101;
C07C 35/30 20130101; C07C 29/76 20130101; C07D 311/40 20130101;
C07C 67/56 20130101; C07C 33/02 20130101; A61P 37/04 20180101; A61P
3/06 20180101; A23V 2002/00 20130101; A61P 35/00 20180101; C07C
47/277 20130101; A61P 17/16 20180101; C07C 33/14 20130101; A61P
9/00 20180101; C07C 31/125 20130101; C07C 47/232 20130101; C07H
17/02 20130101; Y02P 20/54 20151101; A61P 1/02 20180101; A61P 3/10
20180101; C07C 47/27 20130101; A61P 31/18 20180101; A61P 19/10
20180101; A23L 27/12 20160801; C07C 47/54 20130101; A61P 39/06
20180101; C07C 33/32 20130101; C07C 45/78 20130101; C07C 29/76
20130101; C07C 31/125 20130101; C07C 29/76 20130101; C07C 33/32
20130101; C07C 51/47 20130101; C07C 57/44 20130101; C07C 67/56
20130101; C07C 69/618 20130101; C07C 67/56 20130101; C07C 69/007
20130101; A23V 2002/00 20130101; A23V 2200/15 20130101; A23V
2250/708 20130101; A23V 2250/264 20130101; C07C 67/56 20130101;
C07C 69/157 20130101 |
Class at
Publication: |
424/739 ;
426/655; 536/123.1; 536/008; 549/397; 549/403; 549/406; 562/405;
568/425; 568/716; 568/717; 568/840; 585/022 |
International
Class: |
A61K 36/54 20060101
A61K036/54; A23L 1/28 20060101 A23L001/28; A61P 39/06 20060101
A61P039/06; C07C 29/00 20060101 C07C029/00; C07C 39/12 20060101
C07C039/12; C07C 39/18 20060101 C07C039/18; C07C 47/54 20060101
C07C047/54; C07C 63/04 20060101 C07C063/04; C07D 311/04 20060101
C07D311/04; C07H 17/02 20060101 C07H017/02 |
Claims
1. A cinnamon species extract comprising a fraction having a Direct
Analysis in Real Time (DART) mass spectrometry chromatogram of any
of FIGS. 6 to 85.
2. The cinnamon species extract of claim 1, wherein the fraction
comprises a compound selected from the group consisting of
cinnamaldehyde, benzaldehyde, cinnamyl alcohol, trans-cinnamic
acid, cinnamyl acetate, an essential oil, a polyphenol, a
polysaccharide, and combinations thereof.
3. The cinnamon species extract of claim 2, wherein the fraction
comprises cinnamaldehyde in an amount greater than about 2% by
weight.
4. The cinnamon species extract of claim 2, wherein the fraction
comprises cinnamaldehyde in an amount greater than about 5, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%
by weight.
5. The cinnamon species extract of claim 2, wherein the fraction
comprises cinnamaldehyde in an amount from about 65% to about 95%
by weight.
6. The cinnamon species extract of claim 2, wherein the fraction
comprises an essential oil selected from the group consisting of
eugenol, 2'-hydroxycinnamaldehyde, 2-methoxycinnamaldehyde,
2'-benzoxycinnamaldehyde, linalool, 1,8-cineole, alpha-pinene,
beta-pinene, and combinations thereof.
7. The cinnamon species extract of claim 2, wherein the fraction
comprises essential oil in an amount from about 1% to about 5% by
weight.
8. The cinnamon species extract of claim 2, wherein the fraction
comprises a combined amount of cinnamaldehyde and essential oil of
about 5% to about 40% by weight.
9. The cinnamon species extract of claim 2, wherein the fraction
comprises a polyphenol selected from the group consisting of
flavonoid, flavonol glycoside, and combinations thereof.
10. The cinnamon species extract of claim 2, wherein the fraction
comprises a polyphenol in an amount from about 20% to about 70% by
weight.
11. The cinnamon species extract of claim 2, wherein the fraction
comprises cinnamaldehyde at about 6% by weight and a polyphenol at
about 70% by weight.
12. The cinnamon species extract of claim 2, wherein the fraction
comprises cinnamaldehyde at about 40% by weight and a polyphenol at
about 20% by weight.
13. The cinnamon species extract of claim 2, wherein the fraction
comprises a polysaccharide selected from the group consisting of
glucose, arabinose, galactose, rhamnose, xylose uronic acid and
combinations thereof.
14. The cinnamon species extract of claim 2, wherein the fraction
comprises a polysaccharide at about 30% by weight.
15. The cinnamon species extract of claim 9, wherein the flavonoid
is selected from the group consisting of
3-(2-hydroxyphenyl)-propanoic acid,
3-(2-hydroxyphenyl)-O-glycoside, anthocyanidin, epitcatechin,
catechin, methylhydroxychalcone, catechin oligomers, epicatechin
oligomers, oligomeric proanthocyanidins, polymeric
proanthocyanidins, and combinations thereof.
16. The cinnamon species extract of claim 9, wherein the flavonol
glycoside is selected from the group consisting of kaempferitrin,
kaempferol
3-O-Beta-D-glucopyranosyl-(1.fwdarw.4)-alpha-L-rhamnopyranoside,
kaempferol
3-O-beta-D-apiofuranosyl-(1.fwdarw.42)-alpha-L-rhamnopyranoside,
kaempferol
3-O-beta-D-apiofuranosyl-(1.fwdarw.4)-alpha-L-rhamnopyranoside, and
combinations thereof.
17. Food or medicament comprising the cinnamon species extract of
claim 1.
18. A method of preparing a cinnamon extract comprising
sequentially extracting a cinnamon species plant material to yield
an essential oil fraction, a non-tannin polyphenolic fraction and a
polysaccharide fraction by a) extracting cinnamon species plant
material by supercritical carbon dioxide extraction to yield the
essential oil fraction and a first residue; b) extracting cinnamon
species plant material or the first residue from step a) by water
at about 70.degree. C. to about 90.degree. C. extraction and
precipitating the polysaccharide with alcohol to yield the
polysaccharide fraction and a second residue; and c) extracting
cinnamon species plant material, the first residue from step a)
and/or the second residue from step b) with a hydro-alcoholic
solution and purifying the extraction using affinity adsorbent
processes to yield the non-tannin polyphenolic fraction.
19. The method of claim 18, wherein step a) comprises 1) loading in
an extraction vessel ground cinnamon species plant material; 2)
adding carbon dioxide under supercritical conditions; 3) contacting
the ground cinnamon bark and the carbon dioxide for a time; and 4)
collecting an essential oil fraction in a collection vessel.
20. The method of claim 19, wherein supercritical conditions
comprise 60 bars to 800 bars of pressure at 35.degree. C. to
90.degree. C.
21. The method of claim 19, wherein supercritical conditions
comprise 60 bars to 500 bars of pressure at 40.degree. C. to
80.degree. C.
22. The method of claim 19, wherein the time is 30 minutes to 2.5
hours.
23. The method of claim 19, wherein the time is 1 hour.
24. The method of claim 19, wherein a supercritical carbon dioxide
fractional separation system is used for fractionation,
purification, and profiling of the essential oil fraction.
25. The method of claim 18, wherein step b) comprises 1) contacting
ground cinnamon species plant material or the first residue from
step a) with a water for a time sufficient to extract
polysaccharide chemical constituent; and 2) separating and
purifying the solid polysaccharides from the solution by alcohol
precipitation.
26. The method of claim 25, wherein the water is at 70.degree. C.
to 90.degree. C.
27. The method of claim 25, wherein the water is at 80.degree. C.
to 90.degree. C.
28. The method of claim 25, wherein the time is 1-5 hours.
29. The method of claim 25, wherein the time is 2-4 hours.
30. The method of claim 25, wherein the time is 2 hours.
31. The method of claim 25, wherein the alcohol is ethanol.
32. The method of claim 18, wherein step c) comprises: 1)
contacting cinnamon species plant material, the first residue from
step a) and/or the second residue from step b) with hydroalcoholic
solution for a time sufficient to extract polyphenolic chemical
constituents; 2) passing a concentrated alcohol solution of
extracted polyphenolic chemical constituents from the
hydroalcoholic solvent mixture through an affinity adsorbent resin
column wherein the polyphenolic acids are adsorbed; and 3) eluting
the purified non-tannin polyphenolic chemical constituent
fraction(s) from the affinity adsorbent resin leaving the tannin
polyphenolics adsorbed to the affinity adsorbent resin.
33. The method of claim 32, wherein the hydroalcoholic solution
comprises ethanol and water wherein the ethanol concentration is
10-95% by weight.
34. The method of claim 32, wherein the hydroalcoholic solution
comprises ethanol and water wherein the ethanol concentration is
25% by weight.
35. The method of claim 32, wherein step 1) is carried out at
30.degree. C. to 100.degree. C.
36. The method of claim 32, wherein step 1) is carried out at
60.degree. C. to 100.degree. C.
37. The method of claim 32, wherein the time is 1-10 hours.
38. The method of claim 32, wherein the time is 1-5 hours.
39. The method of claim 32, wherein the time is 2 hours.
40. A cinnamon species extract prepared by the method of claim
18.
41. A cinnamon species extract comprising cinnamaldehyde, cinnamic
acid at 1 to 5% by weight of the cinnamaldehyde, methyl cinnamic
acid at 5 to 15% by weight of the cinnamaldehyde, cinnamyl alcohol
at 1 to 5% by weight of the cinnamaldehyde,
.beta.-gualenen/cis-.gamma.-bisababolene at 20 to 30% by weight of
the cinnamaldehyde, and pyrogallol at 1 to 5% by weight of the
cinnamaldehyde.
42. A cinnamon species extract comprising pyrogallol, cinnamic acid
at 80 to 90% by weight of the pyrogallol, methyl cinnamic acid at
85 to 95% by weight of the pyrogallol, coumaric acid at 20 to 30%
by weight of the pyrogallol, homovanillic acid at 15 to 25% by
weight of the pyrogallol, cinnamaldehyde at 85 to 95% by weight of
the pyrogallol, and benzyl benzoate at 10 to 15% by weight of the
pyrogallol.
43. A cinnamon species extract comprising catechin, cinnamic acid
at 5 to 15% by weight of the catechin, methyl cinnamic acid at 5 to
15% by weight of the catechin, coumaric acid at 5 to 15% by weight
of the catechin, ferulic acid at 1 to 10% by weight of the
catechin, 2-methoxyphenol at 1 to 5% by weight of the catechin,
homovanillic acid at 5 to 15% by weight of the catechin, vanillic
acid at 20 to 30% by weight of the catechin, benzaldehyde at 1 to
5% by weight of the catechin, cinnamaldehyde at 35 to 45% by weight
of the catechin, pyrogallol at 85 to 95% by weight of the catechin,
and caffeic acid at to 15% by weight of the catechin.
44. A cinnamon species extract comprising
.beta.-gualenen/cis-.gamma.-bisababolene and cinnamaldehyde at 5 to
15% by weight of the .beta.-gualenen/cis-.gamma.-bisababolene.
45. A cinnamon species extract comprising cinnamaldehyde and
.beta.-gualenen/cis-.gamma.-bisababolene at 10 to 20% by weight of
cinnamaldehyde.
46. A cinnamon species extract comprising cinnamaldehyde,
pyrogallol at 30 to 40% by weight of the cinnamaldehyde, and
catechin/epicatechin at 1 to 10% by weight of cinnamaldehyde.
47. A cinnamon species extract comprising cinnamaldehyde, cinnamic
acid at 1 to 5% by weight of the cinnamaldehyde, methoxy
cinnamaldehyde at 0.5 to 5% by weight of the cinnamaldehyde,
eugenol at 0.1 to 5% by weight of the cinnamaldehyde, p-cymene at 1
to 5% by weight of the cinnamaldehyde, camphor at 0.1 to 5% by
weight of the cinnamaldehyde, carvacrol at 0.5 to 5% by weight of
the cinnamaldehyde, caryophyllene/humulene at 25 to 35% by weight
of the cinnamaldehyde, pyrogallol at 0.1 to 5% of the
cinnamaldehyde, and cinnamyl cinnamate at 40 to 50% by weight of
the cinnamaldehyde.
48. A cinnamon species extract comprising cinnamyl cinnamate,
methoxy cinnamaldehyde at 0.5 to 5% by weight of the cinnamyl
cinnamate, cinnamyl alcohol at 0.1 to 5% by weight of the cinnamyl
cinnamate, p-cymene at 1 to 5% by weight of the cinnamyl cinnamate,
linalool at 0.1 to 5% by weight of the cinnamyl cinnamate, camphor
at 0.1 to 5% by weight of the cinnamyl cinnamate, carvacrol at 0.5
to 5% by weight of the cinnamyl cinnamate, cinnamaldehyde at 70 to
80% by weight of the cinnamyl cinnamate, caryophyllene/humulene at
45 to 55% by weight of the cinnamyl cinnamate, and pyrogallol at
0.1 to 5% of the cinnamyl cinnamate.
49. A cinnamon species extract comprising pyrogallol, cinnamic acid
at 5 to 10% by weight of the pyrogallol, coumaric acid at 60 to 70%
by weight of the pyrogallol, ferulic acid at 1 to 10% of the
pyrogallol, 2-methoxyphenol at 5 to 15% of the pyrogallol, vanillic
acid at 1 to 10% by weight of the pyrogallol, catechin/epicatechin
at 30 to 40% by weight of the pyrogallol, benzaldehyde at 1 to 5%
by weight of the pyrogallol, afzelechin/epiafzelechin at 5 to 15%
by weight of the pyrogallol, resveratrol at 1 to 10% by weight of
the pyrogallol, and vanillin at 1 to 5% by weight of the
pyrogallol.
50. A cinnamon species extract comprising pyrogallol, cinnamic acid
at 0.5 to 5% by weight of the pyrogallol, coumaric acid at 10 to
20% by weight of the pyrogallol, ferulic acid at 0.5 to 5% of the
pyrogallol, 2-methoxyphenol at 1 to 5% of the pyrogallol,
homo/isovanillic acid at 0.5 to 5% by weight of the pyrogallol,
vanillic acid at 1 to 10% by weight of the pyrogallol,
catechin/epicatechin at 25 to 35% by weight of the pyrogallol,
benzaldehyde at 1 to 5% by weight of the pyrogallol, cinnamaldehyde
at 1 to 5% of the pyrogallol, afzelechin/epiafzelechin at 0.1 to 5%
by weight of the pyrogallol, and vanillin at 65 to 75% by weight of
the pyrogallol.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Applications Ser. Nos. 60/785,012, filed Mar.
23, 2006, and 60/873,475, filed Dec. 7, 2006, which are hereby
incorporated by reference in their entirety.
FIELD OF INVENTION
[0002] The disclosure relates in part to extractions derived from
cinnamon species, having an elevated essential oil amount, an
elevated phenolic acid amount, an elevated proanthocyanidin amount,
and/or an elevated polysaccharide amount, methods of preparing such
extractions, and methods for use of such extractions.
BACKGROUND OF THE INVENTION
[0003] Cinnamon (Cinnamomum zeylanicum or verum, C. aromaticum, and
C. cassia) is a small evergreen tree 10-15 meters tall that is
native to tropical southern India and Sri Lanka and grows from sea
level to elevations of nine hundred meters. It has thick scabrous
bark and strong branches. Young shoots are speckled greenish
orange. The leaves are petiolate and leathery when mature, with a
shiny green upper side and lighter underside. The leaves smell
spicy and have a hot taste. The fruit is an oval berry, larger than
a blackberry; like an acorn in its receptacle. The fruit is bluish
when ripe with white spots on it, with a taste like Juniper and a
terebine smell. When boiled, it gives off an oily matter which is
called cinnamon suet. The root-bark smells like cinnamon and tastes
like camphor, which can be isolated via distillation. "cinnamon",
the medicinal part of cinnamonum species, consists of the dried
bark, separated from the cork and the underlying parenchyma, of
young branches and shoots of Cinnamoum species.
[0004] Cinnamon species were introduced throughout the islands of
the Indian Ocean and Southeast Asia, and are now cultivated
extensively in Sri Lanka and the coastal regions of India. Sri
Lanka is the main producing country, though substantial cinnamon
product comes from India, Malaysia, Madagascar and the, Seychelles.
Cinnamon bark has been used in traditional Eastern and Western
medicines for several thousand years. According to the energetics
theory in traditional Chinese medicine (TCM), cinnamon acts to
supplement the body fire, to warm and tone the spleen and kidney;
thus making it effective for chest and abdominal pain, diarrhea due
to asthenia, and hypofunction of the kidney. Galenical preparations
of cinnamon are used as a carminative, digestive, or stomachic
component of compounds in TCM, traditional Greco-European
medicines, and traditional Indian Ayurvedic and Unani medicine. The
German Commission E approved the internal use of cinnamon for loss
of appetite and dyspeptic complaints such as mild spasms of the
gastrointestinal tract, bloating, and flatulence. In the United
States and Germany, cinnamon is used as a carminative and stomachic
component of herbal compounds in dosage forms including aqueous
infusion or decoction, alcoholic fluid extract or tincture, and
essential oil. It also appears as a component of multi-herb cough,
cold, and fever formulas. More recently, scientific evidence has
supported the use of cinnamon for type 2 diabetes
(NIDDM-non-insulin dependent diabetes mellitus), anti-oxidant
activity, anti-platelet adhesive activity, anti-inflammatory
activity, anti-bacterial and fungal activity, and enhancement of
brain function. See Khan A et al. Diabetes Care 26:3215-3218, 2003;
Anderson R A et al. J Agric Food Chem 52:65-70, 2004;
Jarville-Taylor et al. J Am Coll Nutri 20:327-336, 2001; Qin R et
al. Horm Metab Res 36:119-123, 2004; Vespohl E J et al. Phytother
Res 19:203-206, 2005; Lee S H et al Biochem Pharmacol 69:791-9,
2005; Chericoni S et al. J Agric Food Chem 53:4762-4765, 2005; Lin
C C et al. Phytother Res 17:7260730, 2003; Jayaprakasha G K et al.
J Agric Food Chem 51:4344-4348, 2003; Huss U et al. J Nat Prod
65:1517-21, 2002; Nagai H et al. Jpn J Pharmacol 32:813-822, 1982;
Su M J et al. J Biomed Sci 6:376-386, 1999; Shimada Y et al.
Phytomed 11:404-410, 2004; Taher M et al. Med J Malayia 59B:97-98,
2004; Kamath J V et al. Phytother Res 17:970-972, 2003; Kurokawa M
et al. Eur J Pharmacol 348:45-51, 1998; Simic A et al. Phytother
Res 18:713-717, 2004; Tabak M et al. J Ethnopharmacol 67:269-277,
1999; Kong L D et al. J Ethnopharmacol 73:199-207, 2000; Kwon B M
et al. Arch Pharm Res 21:147-152, 1998; Ka H et al. Cancer Lett
196:143-152, 2003.
[0005] The chemical constituents of cinnamon bark include the
essential oils (volatile and non-volatile), polyphenolic acids,
coumarin, gum, muscilage, resin, carbohydrates (starch,
polysaccharides), and ash (Table 1). From a commercial and
biological standpoint, the essential oil (particularly the
cinnamaldehydes and terpenes) and the polyphenolic acids
(particularly the flavonol glycosides-proanthocyanidins and
flavonoids) have been traditionally considered to be of greater
importance than the other constituents. Polyphenolic compounds
contain more than one hydroxyl group (OH) on one or more aromatic
rings. The physical and chemical properties, analysis, and
biological activities of polyphenols and particularly flavonoids
have been studied for many years. However, other chemical
constituents such as the polysaccharides may also have important
biologically beneficial effects. Like all botanicals, the chemical
composition of cinnamon bark varies with species, age of harvest,
climate, soil, and horticultural practices. TABLE-US-00001 TABLE 1
Principal Chemical Constituents of Cinnamon Bark % dry Chemical
constituents weight Essential Oils 1-4% Volatile Oils
Trans-cinnamaldehyde (60-80%) Benzaldehyde 2'-hydroxycinnamaldehyde
2-methoxycinnamaldehyde 2'-benzoxycinnamaldehyde Eugenol (up to
10%) Trans-cinnamic acid (5-10%) Cinnamyl acetate Cinnamyl alcohol
Linalool 1,8-cineole Monoterpenes and Sesquiterpenes (1-3%)
Alpha-Pinene Beta-pinene Borneol Polyphenols 5-10% Flavonol
glycosides Kaempferitrin Kaempferol
3-O-Beta-D-glucopyranosyl-(1.fwdarw.4)-alpha- L-rhamnopyranoside
Kaempferol 3-O-beta-D-apiofuranosyl-(1.fwdarw.2)-alpha-
L-rhamnopyranoside Kaempferol
3-O-beta-D-apiofuranosyl-(1.fwdarw.4)-alpha- L-rhamnopyranoside
Flavonoids Methylhydroxychalcone catechin epicatechin anthocyanidin
Catechin/Epicatechin oligomers 3-(2-hydroxyphenyl)-propanoic acid
3-(2-hydroxyphenyl)-O-glycoside Proanthocyanidins Condensed Tannins
Calcium-monterpenes oxalate Gum Muscilage Resin Carbohydrates
80-90% Starch Polysaccharides Ash
SUMMARY OF THE INVENTION
[0006] In one aspect, the present invention relates to a cinnamon
species extract comprising a fraction having a Direct Analysis in
Real Time (DART) mass spectrometry chromatogram of any of FIGS. 6
to 85.
[0007] In a further embodiment, the fraction comprises a compound
selected from the group consisting of cinnamaldehyde, benzaldehyde,
cinnamyl alcohol, trans-cinnamic acid, cinnamyl acetate, an
essential oil, a polyphenol, a polysaccharide, and combinations
thereof.
[0008] In a further embodiment, the fraction comprises
cinnamaldehyde in an amount greater than about 2% by weight. In a
further embodiment, the fraction comprises cinnamaldehyde in an
amount greater than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, or 95% by weight. In a further
embodiment, the fraction comprises cinnamaldehyde in an amount from
about 65% to about 95% by weight.
[0009] In a further embodiment, the fraction comprises an essential
oil selected from the group consisting of eugenol,
2'-hydroxycinnamaldehyde, 2-methoxycinnamaldehyde,
2'-benzoxycinnamaldehyde, linalool, 1,8-cineole, alpha-pinene,
beta-pinene, and combinations thereof. In a further embodiment, the
fraction comprises essential oil in an amount from about 1% to
about 5% by weight. In a further embodiment, the fraction comprises
a combined amount of cinnamaldehyde and essential oil of about 5%
to about 40% by weight.
[0010] In a further embodiment, the fraction comprises a polyphenol
selected from the group consisting of flavonoid, flavonol
glycoside, and combinations thereof. In a further embodiment, the
flavonoid is selected from the group consisting of
3-(2-hydroxyphenyl)-propanoic acid,
3-(2-hydroxyphenyl)-O-glycoside, anthocyanidin, epitcatechin,
catechin, methylhydroxychalcone, catechin oligomers, epicatechin
oligomers, oligomeric proanthocyanidins, polymeric
proanthocyanidins, and combinations thereof. In a further
embodiment, the flavonol glycoside is selected from the group
consisting of kaempferitrin, kaempferol
3-O-Beta-D-glucopyranosyl-(1.fwdarw.4)-alpha-L-rhamnopyranoside,
kaempferol
3-O-beta-D-apiofuranosyl-(1.fwdarw.2)-alpha-L-rhamnopyranoside,
kaempferol
3-O-beta-D-apiofuranosyl-(1.fwdarw.4)-alpha-L-rhamnopyranoside, and
combinations thereof. In a further embodiment, the fraction
comprises a polyphenol in an amount from about 20% to about 70% by
weight. In a further embodiment, the fraction comprises
cinnamaldehyde at about 6% by weight and a polyphenol at about 70%
by weight. In a further embodiment, the fraction comprises
cinnamaldehyde at about 40% by weight and a polyphenol at about 20%
by weight.
[0011] In a further embodiment, the fraction comprises a
polysaccharide selected from the group consisting of glucose,
arabinose, galactose, rhamnose, xylose uronic acid and combinations
thereof. In a further embodiment, the fraction comprises a
polysaccharide at about 30% by weight.
[0012] In another aspect, the present invention relates to a food
or medicament comprising the cinnamon species extract of the
present invention.
[0013] In another aspect, the present invention relates to a method
for making a cinnamon extract comprising sequentially extracting a
cinnamon species plant material to yield an essential oil fraction,
a non-tannin polyphenolic fraction and a polysaccharide fraction by
a) extracting cinnamon species plant material by supercritical
carbon dioxide extraction to yield the essential oil fraction and a
first residue; b) extracting cinnamon species plant material or the
first residue from step a) with hot water to yield the
polysaccharide fraction and a second residue; and c) extracting
cinnamon species plant material, the first residue from step a)
and/or the second residue from step b) with a hydro-alcoholic
solution and purifying the extraction using affinity adsorbent
processes to yield the non-tannin polyphenolic fraction.
[0014] In a further embodiment, step a) comprises 1) loading in an
extraction vessel ground cinnamon species plant material; 2) adding
carbon dioxide under supercritical conditions; 3) contacting the
ground cinnamon bark and the carbon dioxide for a time; and 4)
collecting an essential oil fraction in a collection vessel. In a
further embodiment, supercritical conditions comprise 60 bars to
800 bars of pressure at 35.degree. C. to 90.degree. C. In a further
embodiment, supercritical conditions comprise 60 bars to 500 bars
of pressure at 40.degree. C. to 80.degree. C. In a further
embodiment, the time is 30 minutes to 2.5 hours. In a further
embodiment, the time is 1 hour. In a further embodiment, a
supercritical carbon dioxide fractional separation system is used
for fractionation, purification, and profiling of the essential oil
fraction.
[0015] In a further embodiment, step b) comprises 1) contacting
ground cinnamon species plant material or the first residue from
step a) with a water solution for a time sufficient to extract
polysaccharide chemical constituent; and 2) separating and
purifying the solid polysaccharides from the solution by alcohol
precipitation. In a further embodiment, the water solution is at
80.degree. C. to 100.degree. C. In a further embodiment, the water
solution is at 80.degree. C. to 90.degree. C. In a further
embodiment, the time is 1-5 hours. In a further embodiment, the
time is 2-4 hours. In a further embodiment, the time is 2 hours. In
a further embodiment, the alcohol is ethanol.
[0016] In a further embodiment, step c) comprises: 1) contacting
cinnamon species plant material, the first residue from step a)
and/or the second residue from step b) with hydroalcoholic solution
for a time sufficient to extract polyphenolic chemical
constituents; 2) passing a concentrated alcohol solution of
extracted polyphenolic chemical constituents from the
hydroalcoholic solvent mixture through an affinity adsorbent resin
column wherein the polyphenolic acids are adsorbed; and 3) eluting
the purified non-tannin polyphenolic chemical constituent
fraction(s) from the affinity adsorbent resin leaving the tannin
polyphenolics adsorbed to the affinity adsorbent resin.
[0017] In a further embodiment, the hydroalcoholic solution
comprises ethanol and water wherein the ethanol concentration is
10-95% by weight. In a further embodiment, the hydroalcoholic
solution comprises ethanol and water wherein the ethanol
concentration is 25% by weight. In a further embodiment, step 1) is
carried out at 30.degree. C. to 100.degree. C. In a further
embodiment, step 1) is carried out at 60.degree. C. to 100.degree.
C. In a further embodiment, the time is 1-10 hours. In a further
embodiment, the time is 1-5 hours. In a further embodiment, the
time is 2 hours.
[0018] In another aspect the present invention relates to a
cinnamon species extract prepared by the methods of the present
invention.
[0019] In another aspect the present invention relates to a
cinnamon species extract comprising cinnamaldehyde, cinnamic acid
at 1 to 5% by weight of the cinnamaldehyde, methyl cinnamic acid at
5 to 15% by weight of the cinnamaldehyde, cinnamyl alcohol at 1 to
5% by weight of the cinnamaldehyde,
.beta.-gualenen/cis-.gamma.-bisababolene at 20 to 30% by weight of
the cinnamaldehyde, and pyrogallol at 1 to 5% by weight of the
cinnamaldehyde.
[0020] In another aspect the present invention relates to a
cinnamon species extract comprising pyrogallol, cinnamic acid at 80
to 90% by weight of the pyrogallol, methyl cinnamic acid at 85 to
95% by weight of the pyrogallol, coumaric acid at 20 to 30% by
weight of the pyrogallol, homovanillic acid at 15 to 25% by weight
of the pyrogallol, cinnamaldehyde at 85 to 95% by weight of the
pyrogallol, and benzyl benzoate at 10 to 15% by weight of the
pyrogallol.
[0021] In another aspect the present invention relates to a
cinnamon species extract comprising catechin, cinnamic acid at 5 to
15% by weight of the catechin, methyl cinnamic acid at 5 to 15% by
weight of the catechin, coumaric acid at 5 to 15% by weight of the
catechin, ferulic acid at 1 to 10% by weight of the catechin,
2-methoxyphenol at 1 to 5% by weight of the catechin, homovanillic
acid at 5 to 15% by weight of the catechin, vanillic acid at 20 to
30% by weight of the catechin, benzaldehyde at 1 to 5% by weight of
the catechin, cinnamaldehyde at 35 to 45% by weight of the
catechin, pyrogallol at 85 to 95% by weight of the catechin, and
caffeic acid at to 15% by weight of the catechin.
[0022] In another aspect the present invention relates to a
cinnamon species extract comprising
.beta.-gualenen/cis-.gamma.-bisababolene and cinnamaldehyde at 5 to
15% by weight of the .beta.-gualenen/cis-.gamma.-bisababolene.
[0023] In another aspect the present invention relates to a
cinnamon species extract comprising cinnamaldehyde and
.beta.-gualenen/cis-.gamma.-bisababolene at 10 to 20% by weight of
cinnamaldehyde.
[0024] In another aspect the present invention relates to a
cinnamon species extract comprising cinnamaldehyde, pyrogallol at
30 to 40% by weight of the cinnamaldehyde, and catechin/epicatechin
at 1 to 10% by weight of cinnamaldehyde.
[0025] In another aspect the present invention relates to a
cinnamon species extract comprising cinnamaldehyde, cinnamic acid
at 1 to 5% by weight of the cinnamaldehyde, methoxy cinnamaldehyde
at 0.5 to 5% by weight of the cinnamaldehyde, eugenol at 0.1 to 5%
by weight of the cinnamaldehyde, p-cymene at 1 to 5% by weight of
the cinnamaldehyde, camphor at 0.1 to 5% by weight of the
cinnamaldehyde, carvacrol at 0.5 to 5% by weight of the
cinnamaldehyde, caryophyllene/humulene at 25 to 35% by weight of
the cinnamaldehyde, pyrogallol at 0.1 to 5% of the cinnamaldehyde,
and cinnamyl cinnamate at 40 to 50% by weight of the
cinnamaldehyde.
[0026] In another aspect the present invention relates to a
cinnamon species extract comprising cinnamyl cinnamate, methoxy
cinnamaldehyde at 0.5 to 5% by weight of the cinnamyl cinnamate,
cinnamyl alcohol at 0.1 to 5% by weight of the cinnamyl cinnamate,
p-cymene at 1 to 5% by weight of the cinnamyl cinnamate, linalool
at 0.1 to 5% by weight of the cinnamyl cinnamate, camphor at 0.1 to
5% by weight of the cinnamyl cinnamate, carvacrol at 0.5 to 5% by
weight of the cinnamyl cinnamate, cinnamaldehyde at 70 to 80% by
weight of the cinnamyl cinnamate, caryophyllene/humulene at 45 to
55% by weight of the cinnamyl cinnamate, and pyrogallol at 0.1 to
5% of the cinnamyl cinnamate.
[0027] In another aspect the present invention relates to a
cinnamon species extract comprising pyrogallol, cinnamic acid at 5
to 10% by weight of the pyrogallol, coumaric acid at 60 to 70% by
weight of the pyrogallol, ferulic acid at 1 to 10% of the
pyrogallol, 2-methoxyphenol at 5 to 15% of the pyrogallol, vanillic
acid at 1 to 10% by weight of the pyrogallol, catechin/epicatechin
at 30 to 40% by weight of the pyrogallol, benzaldehyde at 1 to 5%
by weight of the pyrogallol, afzelechin/epiafzelechin at 5 to 15%
by weight of the pyrogallol, resveratrol at 1 to 10% by weight of
the pyrogallol, and vanillin at 1 to 5% by weight of the
pyrogallol.
[0028] In another aspect the present invention relates to a
cinnamon species extract comprising pyrogallol, cinnamic acid at
0.5 to 5% by weight of the pyrogallol, coumaric acid at 10 to 20%
by weight of the pyrogallol, ferulic acid at 0.5 to 5% of the
pyrogallol, 2-methoxyphenol at 1 to 5% of the pyrogallol,
homo/isovanillic acid at 0.5 to 5% by weight of the pyrogallol,
vanillic acid at 1 to 10% by weight of the pyrogallol,
catechin/epicatechin at 25 to 35% by weight of the pyrogallol,
benzaldehyde at 1 to 5% by weight of the pyrogallol, cinnamaldehyde
at 1 to 5% of the pyrogallol, afzelechin/epiafzelechin at 0.1 to 5%
by weight of the pyrogallol, and vanillin at 65 to 75% by weight of
the pyrogallol.
[0029] The extractions of the disclosure are useful in providing
physiological and medical effects including, but not limited to,
anti-oxidant activity, oxygen free radical scavenging, nitrosation
inhibition, anti-mutagenic activity (cancer prevention),
anti-carcinogenic activity (cancer therapy), skin protection,
anti-aging, anti-cardiovascular disease, anti-stroke disease and
therapy, cerebral protection, anti-hyperlipidemia, anti-periodontal
disease, anti-osteoporosis, immunological enhancement, anti-viral,
anti-HIV and anti-bacterial activity, anti-fungal activity,
anti-viral activity, weight control and thermogenesis,
anti-diabetes, and anxiety reduction, mood enhancement and
cognitive enhancement
[0030] These embodiments of the disclosure, other embodiments, and
their features and characteristics, will be apparent from the
description, drawings and claims that follow.
BRIEF DESCRIPTION OF THE INVENTION
[0031] FIG. 1 depicts an exemplary schematic diagram of cinnamon
extraction processes
[0032] FIG. 2 depicts an exemplary method for the preparation of
essential oil fractions.
[0033] FIG. 3 depicts an exemplary method for preparation of
polysaccharide fractions.
[0034] FIG. 4 depicts an exemplary method for solvent leaching
extraction.
[0035] FIG. 5 depicts an exemplary method for preparation of
purified polyphenolic fractions.
[0036] FIG. 6 depicts AccuTOF-DART Mass Spectrum for cinnamon
polysaccharide (positive ion mode).
[0037] FIG. 7 depicts AccuTOF-DART Mass Spectrum for cinnamon
polysaccharide (negative ion mode).
[0038] FIG. 8 depicts AccuTOF-DART Mass Spectrum for cinnamon bark
(positive ion mode).
[0039] FIG. 9 depicts AccuTOF-DART Mass Spectrum for crude extract
of cinnamon bark separated by column chromatography using Sephadex
LH-20 packing material (positive ion mode).
[0040] FIG. 10 depicts AccuTOF-DART Mass Spectrum for crude extract
of cinnamon bark HS#147 using a 75% EtOH extraction solvent
(positive ion mode).
[0041] FIG. 11 depicts AccuTOF-DART Mass Spectrum for fraction F3
separated by column chromatography using Sephadex LH-20 packing
material (positive ion mode).
[0042] FIG. 12 depicts AccuTOF-DART Mass Spectrum for fraction F4
by column chromatography using Sephadex LH-20 packing material
(positive ion mode).
[0043] FIG. 13 depicts AccuTOF-DART Mass Spectrum for fraction F5
by column chromatography using Sephadex LH-20 packing material
(positive ion mode).
[0044] FIG. 14 depicts AccuTOF-DART Mass Spectrum for fraction F6
by column chromatography using Sephadex LH-20 packing material
(positive ion mode).
[0045] FIG. 15 depicts AccuTOF-DART Mass Spectrum for fraction F7
by column chromatography using Sephadex LH-20 packing material
(positive ion mode).
[0046] FIG. 16 depicts AccuTOF-DART Mass Spectrum for fraction F8
by column chromatography using Sephadex LH-20 packing material
(positive ion mode).
[0047] FIG. 17 depicts AccuTOF-DART Mass Spectrum for cinnamon bark
(negative ion mode).
[0048] FIG. 18 depicts AccuTOF-DART Mass Spectrum for crude extract
of cinnamon bark HS#147 using a 75% EtOH extraction solvent
(negative ion mode).
[0049] FIG. 19 depicts AccuTOF-DART Mass Spectrum for crude extract
of cinnamon bark separated by column chromatography using Sephadex
LH-20 packing material (negative ion mode).
[0050] FIG. 20 depicts AccuTOF-DART Mass Spectrum for fraction F3
separated by column chromatography using Sephadex LH-20 packing
material (negative ion mode).
[0051] FIG. 21 depicts AccuTOF-DART Mass Spectrum for fraction F4
by column chromatography using Sephadex LH-20 packing material
(negative ion mode).
[0052] FIG. 22 depicts AccuTOF-DART Mass Spectrum for fraction F5
by column chromatography using Sephadex LH-20 packing material
(negative ion mode).
[0053] FIG. 23 depicts AccuTOF-DART Mass Spectrum for fraction F6
by column chromatography using Sephadex LH-20 packing material
(negative ion mode).
[0054] FIG. 24 depicts AccuTOF-DART Mass Spectrum for fraction F7
by column chromatography using Sephadex LH-20 packing material
(negative ion mode).
[0055] FIG. 25 depicts AccuTOF-DART Mass Spectrum for fraction F8
by column chromatography using Sephadex LH-20 packing material
(negative ion mode).
[0056] FIG. 26 depicts AccuTOF-DART Mass Spectrum for cinnamon
stick purchased commercially from Mountain Rose Herbs (positive ion
mode).
[0057] FIG. 27 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 40.degree. C. and
100 bar (positive ion mode).
[0058] FIG. 28 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 40.degree. C. and
300 bar (positive ion mode).
[0059] FIG. 29 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 40.degree. C. and
500 bar (positive ion mode).
[0060] FIG. 30 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 60.degree. C. and
100 bar (positive ion mode).
[0061] FIG. 31 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 60.degree. C. and
300 bar (positive ion mode).
[0062] FIG. 32 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 60.degree. C. and
500 bar (positive ion mode).
[0063] FIG. 33 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 80.degree. C. and
100 bar (positive ion mode).
[0064] FIG. 34 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 80.degree. C. and
300 bar (positive ion mode).
[0065] FIG. 35 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 80.degree. C. and
500 bar (positive ion mode).
[0066] FIG. 36 depicts AccuTOF-DART Mass Spectrum for 80% EtOH
leaching extract of crude cinnamon (positive ion mode).
[0067] FIG. 37 depicts AccuTOF-DART Mass Spectrum for 80% EtOH
leaching extract of residue from SCCO.sub.2 extraction of crude
cinnamon (positive ion mode).
[0068] FIG. 38 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F4 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (positive ion mode).
[0069] FIG. 39 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F5 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (positive ion mode).
[0070] FIG. 40 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F6 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (positive ion mode).
[0071] FIG. 41 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F7 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (positive ion mode).
[0072] FIG. 42 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F8 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (positive ion mode).
[0073] FIG. 43 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F9 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (positive ion mode).
[0074] FIG. 44 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F10 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (positive ion mode).
[0075] FIG. 45 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F11 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (positive ion mode).
[0076] FIG. 46 depicts AccuTOF-DART Mass Spectrum for cinnamon
crude extract from HS114 (positive ion mode).
[0077] FIG. 47 depicts AccuTOF-DART Mass Spectrum for cinnamon
crude extract from HS114 (SCCO.sub.2) (positive ion mode).
[0078] FIG. 48 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F4 after thiolytic degradation from
Sepadex LH-20 (positive ion mode).
[0079] FIG. 49 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F5 after thiolytic degradation from
Sepadex LH-20 (positive ion mode).
[0080] FIG. 50 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F6 after thiolytic degradation from
Sepadex LH-20 (positive ion mode).
[0081] FIG. 51 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F7 after thiolytic degradation from
Sepadex LH-20 (positive ion mode).
[0082] FIG. 52 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F8 after thiolytic degradation from
Sepadex LH-20 (positive ion mode).
[0083] FIG. 53 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F9 after thiolytic degradation from
Sepadex LH-20 (positive ion mode).
[0084] FIG. 54 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F10 after thiolytic degradation from
Sepadex LH-20 (positive ion mode).
[0085] FIG. 55 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F11 after thiolytic degradation from
Sepadex LH-20 (positive ion mode).
[0086] FIG. 56 depicts AccuTOF-DART Mass Spectrum for cinnamon
stick purchased commercially from Mountain Rose Herbs (negative ion
mode).
[0087] FIG. 57 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 40.degree. C. and
100 bar (negative ion mode).
[0088] FIG. 58 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 40.degree. C. and
300 bar (negative ion mode).
[0089] FIG. 59 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 40.degree. C. and
500 bar (negative ion mode).
[0090] FIG. 60 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 60.degree. C. and
100 bar (negative ion mode).
[0091] FIG. 61 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 60.degree. C. and
300 bar (negative ion mode).
[0092] FIG. 62 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 60.degree. C. and
500 bar (negative ion mode).
[0093] FIG. 63 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 80.degree. C. and
100 bar (negative ion mode).
[0094] FIG. 64 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 80.degree. C. and
300 bar (negative ion mode).
[0095] FIG. 65 depicts AccuTOF-DART Mass Spectrum for cinnamon
essential oil extracted by SCCO.sub.2 methods at 80.degree. C. and
500 bar (negative ion mode).
[0096] FIG. 66 depicts AccuTOF-DART Mass Spectrum for 80% EtOH
leaching extract of crude cinnamon (negative ion mode).
[0097] FIG. 67 depicts AccuTOF-DART Mass Spectrum for 80% EtOH
leaching extract of residue from SCCO.sub.2 extraction of crude
cinnamon (negative ion mode).
[0098] FIG. 68 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F4 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (negative ion mode).
[0099] FIG. 69 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F5 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (negative ion mode).
[0100] FIG. 70 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F6 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (negative ion mode).
[0101] FIG. 71 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F7 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (negative ion mode).
[0102] FIG. 72 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F8 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (negative ion mode).
[0103] FIG. 73 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F9 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (negative ion mode).
[0104] FIG. 74 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F10 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (negative ion mode).
[0105] FIG. 75 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F11 using Sephadex LH-20 packing material
of HS114 SCCO.sub.2 residue (negative ion mode).
[0106] FIG. 76 depicts AccuTOF-DART Mass Spectrum for cinnamon
crude extract from HS114 (negative ion mode).
[0107] FIG. 77 depicts AccuTOF-DART Mass Spectrum for cinnamon
crude extract from HS114 (SCCO.sub.2) (negative ion mode).
[0108] FIG. 78 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F4 after thiolytic degradation from
Sepadex LH-20 (negative ion mode).
[0109] FIG. 79 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F5 after thiolytic degradation from
Sepadex LH-20 (negative ion mode).
[0110] FIG. 80 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F6 after thiolytic degradation from
Sepadex LH-20 (negative ion mode).
[0111] FIG. 81 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F7 after thiolytic degradation from
Sepadex LH-20 (negative ion mode).
[0112] FIG. 82 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F8 after thiolytic degradation from
Sepadex LH-20 (negative ion mode).
[0113] FIG. 83 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F9 after thiolytic degradation from
Sepadex LH-20 (negative ion mode).
[0114] FIG. 84 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F10 after thiolytic degradation from
Sepadex LH-20 (negative ion mode).
[0115] FIG. 85 depicts AccuTOF-DART Mass Spectrum for cinnamon
ethanol elution fraction F11 after thiolytic degradation from
Sepadex LH-20 (negative ion mode).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0116] As used herein, cinnamon refers to the bark plant material
derived from the Cinnamomum species botanical. The term "cinnamon"
is also used interchangeably with cinnamon species and relates to
said plants, clones, variants, and sports, etc.
[0117] As used herein, the term "one or more compounds" means that
at least one compound, such as, but not limited to,
trans-cinnamaldehyde (a lipid soluble essential oil chemical
constituent of cinnamon species), or methylhydroxychalcone (a water
soluble polyphenolic of cinnamon species) or a polysaccharide
molecule of cinnamon species is intended, or that more than one
compound, for example, trans-cinnamaldehyde and
methylhydroxychalcone is intended.
[0118] As used herein, the term "fraction" means the extraction
comprising a specific group of chemical compounds characterized by
certain physical and/or chemical properties.
[0119] As used herein, the term "essential oil fraction" refers to
a fraction comprising lipid soluble, water insoluble compounds
obtained or derived from cinnamon and related species including,
but not limited to, the chemical compound classified as
trans-cinnamaldehyde.
[0120] As used herein, the term "essential oil sub-fraction" refers
to a fraction comprising lipid soluble, water insoluble compounds
obtained or derived from cinnamon and related species including,
but not limited to, the chemical compound classified as
trans-cinnamaldehyde having enhanced concentrations of specific
compounds found in the essential oil of cinnamon species.
[0121] As used herein, the term "polyphenolic fraction" refers to a
fraction comprising the water soluble and ethanol soluble
polyphenolic acid compounds obtained or derived from cinnamon and
related species, further comprising, but not limited to, compounds
such as methylhydroxychalcone, and catechin and epicatechin
oligomers.
[0122] As used herein, the term "polysaccharide fraction" refers to
a fraction comprising soluble-ethanol insoluble polysaccharide
compounds obtained or derived from cinnamon and related
species.
[0123] Other chemical constituents of cinnamon may also be present
in these extraction fractions.
[0124] As used herein, the term "purified" fraction relates to a
fraction comprising a specific group of compounds characterized by
certain physical-chemical properties or physical or chemical
properties that are concentrated to greater than 20% of the
fraction's chemical constituents. In other words, a purified
fraction comprises less than 80% chemical constituent compounds
that are not characterized by certain desired physical-chemical
properties or physical or chemical properties that define the
fraction.
[0125] As used herein, the term "profile" refers to the ratios by
percent mass weight of the chemical compounds within an extraction
fraction or sub-fraction or to the ratios of the percent mass
weight of each of the three cinnamon fraction chemical constituents
in a final cinnamon extraction.
[0126] As used herein, "feedstock" generally refers to raw plant
material, comprising whole plants alone, or in combination with on
or more constituent parts of a plant comprising leaves, roots,
including, but not limited to, main roots, tail roots, and fiber
roots, stems, bark, leaves, seeds, and flowers, wherein the plant
or constituent parts may comprise material that is raw, dried,
steamed, heated or otherwise subjected to physical processing to
facilitate processing, which may further comprise material that is
intact, chopped, diced, milled, ground or otherwise processed to
affected the size and physical integrity of the plant material.
Occasionally, the term "feedstock" may be used to characterize an
extraction product that is to be used as feed source for additional
extraction processes.
[0127] As used herein, the term "cinnamon constituents" shall mean
chemical compounds found in cinnamon species and shall include all
such chemical compounds identified above as well as other compounds
found in cinnamon species, including but not limited to the
essential oil chemical constituents, polyphenolic acids, and
polysaccharides.
[0128] The chemical constituents of cinnamon are of extensive
therapeutic value. Recent scientific research and clinical studies
have demonstrated the following therapeutic effects of the various
chemical compounds, chemical fractions, and gross extraction
products of cinnamon which include the following: NIDDM-type 2
diabetes mellitus (proanthocyanidins, methylhydroxychalcone,
catechins and epicatechin oligomers, flavonoids, water soluble
extract); Improved cholesterol metabolism including decreased low
density lipoprotein (phenolic acids including proanthocyanidins,
methylhydroxychacone, catechins, epicatechin oigomers, flavonoids,
water soluble extract); anti-artery damaging free radicals and
improved function of small blood vessels (essential oils,
cinnamaldehyde, 2'-hydroxycinnamaldehyde, 2'-methoxycinnmaldehyde,
phenolic acids, flavonoids glycosides, proanthocyanidins,
flavonoids, catechins, epicatechin oligomers, extract);
anti-thrombotic and anti-platelet aggregation (essential oil,
cinnamaldehyde); anti-inflammatory activity (essential oil,
cinnamaldehyde, eugenol, 1,8 cineole, alpha-pinene, beta-pinene,
borneol, flavonol glycosides, extract); anti-oxidant (phenolic
acids, flavonol glycosides, proanthocyanidins, flavonoids, water
soluble extract); anti-allergic (phenolic acids, flavonol
glycosides, proanthocyanidins, flavonoids, water soluble extract);
Neurological protectant (water soluble extract); cardiovascular
protectant (essential oil, water soluble extract); enhanced brain
function (essential oil, particularly volatile oils); caminative,
loss of appetite, dyspeptive complaints, anti-vomiting,
anti-bloating & flatulence, promotion of intestinal motility,
facilitation of weight gain, (flavonoids,
3-(2-hydroxyphenyl)-propanoic acid,
3-(2-hydroxyphenyl)-O-glycoside, water soluble extract);
anti-cough, common cold and fever (essential oil, cinnamyl
acetate); anti-bacterial & anti-fungal activity (essential oil,
cinnamaldehyde, eugenol, 1,8-cineole, beta-pinene, borneol);
lipolytic & improved wound healing (ethanol extract); and
anti-cancer & anti-gout (essential oil, cinnamaldehyde,
2'-hydroxycinnamaldehyde, 2'-benzoxycinnamaldehyde, methanol
extract); See Khan A et al. Diabetes Care 26:3215-3218, 2003;
Anderson R A et al. J Agric Food Chem 52:65-70, 2004;
Jarville-Taylor et al. J Am Coll Nutri 20:327-336, 2001; Qin R et
al. Horm Metab Res 36:119-123, 2004; Vespohl E J et al. Phytother
Res 19:203-206, 2005; Lee S H et al Biochem Pharmacol 69:791-9,
2005; Chericoni S et al. J Agric Food Chem 53:4762-4765, 2005; Lin
C C et al. Phytother Res 17:7260730, 2003; Jayaprakasha G K et al.
J Agric Food Chem 51:4344-4348, 2003; Huss U et al. J Nat Prod
65:1517-21, 2002; Nagai H et al. Jpn J Pharmacol 32:813-822, 1982;
Su M J et al. J Biomed Sci 6:376-386, 1999; Shimada Y et al.
Phytomed 11:404-410, 2004; Taher M et al. Med J Malayia 59B:97-98,
2004; Kamath J V et al. Phytother Res 17:970-972, 2003; Kurokawa M
et al. Eur J Pharmacol 348:45-51, 1998; Simic A et al. Phytother
Res 18:713-717, 2004; Tabak M et al. J Ethnopharmacol 67:269-277,
1999; Kong L D et al. J Ethnopharmacol 73:199-207, 2000; Kwon B M
et al. Arch Pharm Res 21:147-152, 1998; Ka H et al. Cancer Lett
196:143-152, 2003.
[0129] Anthocyanins are a particular class of naturally occurring
flavonoid compounds that are responsible for the red, purple, and
blue colors of many fruits, vegetables, cereal grains, and flowers.
For example, the colors of fruits such as blueberries, bilberries,
strawberries, raspberries, boysenberries, marionberries,
cranberries, elderberries, etc. are due to many different
anthocyanins. Recently, the interest in anthocyanin pigments has
intensified because of their possible health benefits as dietary
antioxidants. For example, anthocyanin pigments of bilberries
(Vaccinium myrtillus) have long been used for improving visual
acuity and treating circulatory disorders. There is experimental
evidence that certain anthocyanins and other flavonoids have
anti-inflammatory properties. In addition, there are reports that
orally administered anthocyanins are beneficial for treating
diabetes and ulcers and may have antiviral and antimicrobial
activities. The chemical basis for these desirable properties of
flavonoids is believed to be related to their antioxidant capacity.
Thus, the antioxidant characteristics associated with berries and
other fruits and vegetables have been attributed to their
anthocyanin content.
[0130] Proanthocyanidins, also known as "oligomeric
proanthocyanidins," "OPCs," or "procyanidins," are another class of
naturally occurring flavonoid compounds widely available in fruits,
vegetables, nuts, seeds, flowers, and barks. Proanthocyanidins
belong to the category known as condensed tannins. They are the
most common type of tannins found in fruits and vegetables, and are
present in large quantities in the seeds and skins. In nature,
mixtures of different proanthocyanidins are commonly found
together, ranging from individual units to complex molecules
(oligomers or polymers) of many linked units. The general chemical
structure of a polymeric proanthocyanidin comprises linear chains
of flavonoid 3-ol units linked together through common C(4)-C(6)
and/or C(4)-C(8) bonds. The proanthocyanidins are mixtures of
oligomers and polymers containing catechin and/or epicatechin units
linked through C4-C8 and/or C4-C6 bonds. These flavan-3-ols can
also be doubly linked by a C4-C8 bond and an additional ether bond
between C7-C2. .sup.13C NMR has been useful in identifying the
structures of polymeric proanthocyanidins, and recent work has
elucidated the chemistry of di-, tri-, and tetrameric
proanthocyanidins. Larger oligomers of the flavonoid 3-ol units are
predominant in most plants and are found with average molecular
weights above 2,000 Daltons and containing 6 or more monomer units.
(Newman, et al., Mag. Res. Chem., 25:118 (1987)). Considerable
recent research has explored the therapeutic applications of
proanthocyanidins, which are primarily known for their antioxidant
activity. However, these compounds have also been reported to
demonstrate antibacterial, antiviral, anticarcinogenic,
anti-inflammatory, anti-allergic, and vasodilatory actions. In
addition, they have been found to inhibit lipid peroxidation,
platelet aggregation, capillary permeability and fragility, and to
affect enzyme systems including phospholipase A2, cyclooxygenase,
and lipoxygenase. For example, proanthocyanidin monomers (i.e.,
anthocyanins) and dimers have been used in the treatment of
diseases associated with increased capillary fragility and have
also been shown to have anti-inflammatory effects in animals
(Beladi, I. et al., Ann. N.Y. Acad. Sci., 284:358 (1977)). Based on
these reported findings, oligomeric proanthocyanidins (OPCs) may be
useful components in the treatment of a number of conditions (Fine,
A. M., Altern. Med. Rev. 5(2):144-151 (2000)).
[0131] Proanthocyanidins may also protect against viruses. In in
vitro studies, proanthocyanidins from witch hazel (Hamamelis
virginiana) killed the Herpes simplex 1 (HSV-1) virus (Erdelmeier,
C. A., Cinatl, J., Plant Med. June: 62(3):241-5 (1996); DeBruyne,
T., Pieters, L., J. Nat. Prod. July: 62(7):954-8 (1999)). Another
study was carried out to determine the structure-activity
relationships of the antiviral activity of various tannins. It was
found that the more condensed the chemical structure, the greater
the antiviral effect (Takechi, M., et al., Phytochemistry,
24:2245-50 (1985)). In another study, proanthocyanidins were shown
to have anti-Herpes simplex activity in which the 50 percent
effective doses needed to reduce herpes simplex plaque formation
were two to three orders of magnitude less than the 50 percent
cytotoxic doses (Fukuchi, K., et al., Antiviral Res., 11:285-298
(1989)).
[0132] Cyclooxygenase (COX-1, COX-2) or prostaglandin endoperoxide
H synthase (PGHS-1, PGHS-2) enzymes are widely used to measure the
anti-inflammatory effects of plant products (Bayer, T., et al.,
Phytochemistry, 28:2373-2378 (1989); and Goda, Y., et al., Chem.
Pharm. Bull., 40:2452-2457 (1992)). COX enzymes are the
pharmacological target sites for nonsteroidal anti-inflammatory
drugs (Humes, J. L., et al., Proc. Natl. Acad. Sci. U.S.A.,
78:2053-2056 (1981); and Rome, L. H., et al., Proc. Natl. Acad.
Sci. U.S.A., 72:4863-4865 (1975)). Two isozymes of cyclooxygenase
involved in prostaglandin synthesis are cyclooxygenase-1 (COX-1)
and cyclooxygenase-2 (COX-2) (Hemler, M., et al., J. Biol. Chem.,
25:251, 5575-5579 (1976)). It is hypothesized that selective COX-2
inhibitors are mainly responsible for anti-inflammatory activity
(Masferrer, J. L., et al., Proc. Natl. Acad. Sci. U.S.A.,
91:3228-3232 (1994)). Flavonoids are now being investigated as
anti-inflammatory substances, as well as for their structural
features for cyclooxygenase (COX) inhibition activity.
[0133] Although cinnamon is generally safe and non-toxic even at
high doses, it may induce allergic reactions in individuals who are
sensitive to cinnamon or Peruvian balsa. It is not recommended
during pregnancy and lactation. There are no known interactions
with other drugs.
[0134] What is needed are novel and reproducible cinnamon extracts
that combine purified essential oil, purified polyphenolics with
high flavonol glycosides and flavonoids, and polysaccharide
chemical constituent fractions that can be produced with
standardized and reliable amounts of these synergistically acting,
physiologically and medically beneficial cinnamon chemical
constituents. Williamson E M. Phtomedicine 8:401-409, 2001.
Extractions
[0135] Essential Oil Fraction
[0136] Cinnamon bark is rich in essential oil and provides various
kinds of oils depending on the part of plant used. It was reported
that there is 1-2% essential oil by % mass weight in cinnamon bark.
The main component of cinnamon bark oil is the aromatic
aldehyde-3-phenyl-2(E)-propenal, also called cinnamaldehyde (about
60% in essential oil by mass weight).
[0137] Cinnamon bark was used as feedstock for current research.
Supercritical carbon dioxide extraction and fractionation
technology has been chosen for extraction due to its well-known
benefit on processing of lipid soluble chemicals. Its usefulness
for extraction is due to the combination of gas-like mass transfer
properties and liquid-like solvating characteristics with diffusion
coefficients greater than those of liquid solvents. The extracted
essential oil constituents were assayed using gas
chromatography-mass spectroscopy. Total 71 compounds have been
identified from cinnamon bark oil extracted by supercritical CO2.
Besides major cinnamaldehyde's congeners, such as benzaldehyde
(P1), cinnamaldehyde (P10 and P14), cinnamyl alcohol (P16),
trans-cinnamic acid (P23), cinnamyl acetate (P25), other minor
compounds including: 4 monoterpenes, 16 sesquiterpenes, 9 fatty
acids and their derivatives, and 6 steroids (P64 and P67 P71) have
also been identified. Fatty acids and steroids have not previously
been reported in cinnamon oil.
[0138] It was found that supercritical CO2 is an excellent tool to
purify and profile essential oil fractions. The extraction yield of
these fractions varies depending on processing temperature,
pressure, and solvent/feed ratio. The highest extraction yield was
1.76% by mass weight at temperature of 80.degree. C. and pressure
of 100-500 bar with a solvent/feed ratio of 114. In crude extracted
cinnamon bark essential oil, cinnamaldehyde accounts for 58%-69% by
mass weight of the purified fractions. It was found that up to 20%
of steroid compounds in extracts in extract fractions can only be
extracted at low temperatures of about 40 C. High purity of
cinnamaldehyde's congeners (greater than 90%) can be obtained at
high temperatures of 60-90.degree. C. and low pressures of about
100 bar. High pressure and temperature are better for processing
fatty acid compounds and the highest purity can be up to .about.10%
in extract fractions.
[0139] The crude extracted cinnamon bark essential oil can also be
fractioned by multistage stage processing by increase processing
pressure sequentially at fixed temperature. The results are shown
in Table 2. It was found that the major compounds cinnamaldehyde
congeners can be profiled between 67.1-93.1%. Other minor
compounds, as sesquiterpene can be profiled between 1.1-2.7%; fatty
acid can be profiled between 0.9-9.9%; steroids can only be
extracted at temperature of 40.degree. C. and can be profiled
between 0.0-20.3% by % mass weight of the fraction (relative
abundance). The highest purity of cinnamaldehyde can be up to
91.13%, which is 76 times greater than that found of that in
cinnamon bark feedstock. TABLE-US-00002 TABLE 2 Cinnamon essential
oil compounds profile in extracts obtained at different conditions
T = 40.degree. C. T = 60.degree. C. T = 80.degree. C. Compounds
Stage 1 Stage 2 Stage 3 Stage 4 Stage 1 Stage 2 Stage 3 Stage 1
Stage 2 Stage 3 Cinnamaldehyde 67.3 88.0 83.3 67.1 93.1 86.2 74.7
90.7 88.9 74.1 congeners Sesquiterpene 1.4 1.5 2.1 2.1 2.7 1.7 2.0
1.1 1.1 3.5 Fatty acids and 0.9 2.5 6.6 9.9 0.9 5.9 8.6 1.0 4.1 7.8
derivatives Steroids 20.3 5.2 0.3 0.8 0.0 0.0 0.0 0.0 0.0 0.0
[0140] Phenolic Acid Fraction
[0141] Antioxidant activity of cinnamon is related to the phenolic
acid chemical constituent content. Specific antioxidant
phytochemicals that have been identified in cinnamon include the
following phenolic acids: epicatechin, camphene, engenol,
gamma-terpinene, phenol, salicylic acid and tannins. More recently,
scientists at the US department of agriculture found one type of
flavonoid, type-A procyanidin, extracted by water that mimics the
effect of insulin. This compound potentiates insulin action in
isolated adipocytes. In-vivo studies also showed that cinnamon
water extracts improve insulin actions via increasing glucose
uptake, in part through enhancing the insulin-signaling pathway in
skeletal muscle. The object of this section of the present
invention is to purify phenolic acids by removing tannin acids. The
phenolic acids of interests due to their hypoglycemic activity are
the proanthocyanidins. The proanthocyanindins are mixtures of
oligomers and polymers containing (+)-catechin and/or
(-)-epicatechin units linked through C4-C8 and/or C4-C6 bonds
(B-type). These flavan-3-ol can also be doubled linked by a C4-C8
bond and an additional ether bond between C7-C2 bond (A-type). Due
to lack of a commercial available HPLC reference standard, the
Folin-Ciocalteu method was used to analysis total phenolic acid
content and the protein-precipitable phenolics method to analysis
total tannin acid content. Individual phenolic acids in the total
phenolic acids were identified and semi-quantified by Direct
Analysis in Real-time (DART) mass spectrometry.
[0142] In the cinnamon bark feedstock, there is about 4.87% total
phenolic acid, in which about 2.27% is nontannin phenolic acids and
about 2.61% is tannin acids. Total phenolic acid extraction
conditions were optimized by studying the effect of different
solvents, temperatures, PH values, and multistage processing. It
was found that aqueous ethanol (25-75% ethanol) were optimum
extraction solvents. The highest extraction yield were found at
about 17.6% by using 25% ethanol as the extraction solvent at a
temperature of 40.degree. C. using two stage of processing at
solvent feed ratio of 10 and 5 respectively. No pH value change
needed during processing.
[0143] Sephadex LH-20 dextran beads were found to be an excellent
media to separate nontannin phenolic acids from tannin acids. The
results are shown in Table 3. It was found that tannin acid has
been remarkably removed and nontannin phenolic acid has been
purified to up to 100% (44 fold of that in feedstock).
TABLE-US-00003 TABLE 3 Cinnamon phenolics weight percentage
changing during sephadex LH-20 processing. B1- B1- Feed B1-F3 B1-F4
B1-F5 B1-F6 F7 F8 Weight % Nontannin 2.27 29.5 66.4 87.8 91.1 100
93.8 phenolic acids Tannin acids 2.61 0 0 0 0 0 0
[0144] Polysaccharide Fraction
[0145] Cinnamon polysaccharide-glycoprotein fraction were obtained
by water extraction and 80% ethanol precipitation. The yield of
purified cinnamon polysaccharide-glycoprotein fractions was about
3.5%. The purity of cinnamon polysaccharide was 0.29-0.47 g dextran
equivalent/g polysaccharide. (Dextran was used as reference
standard because no cinnamon polysaccharide standards are
available). The average molecular weight of cinnamon polysaccharide
was .about.2500 KDa. AccuTOF-DART mass spectrometry was also used
to characterize cinnamon polysaccharide, the results are shown in
FIGS. 6 and 7.
Extractions Relative to Natural Cinnamon Species
[0146] This disclosure comprises extractions of isolated and
purified fractions of essential oils (or essential oil
sub-fractions), polyphenolic acids, and polysaccharides from one or
more cinnamon species. These individual fractions can be combined
in specific ratios (profiles) to provide beneficial combinations
and can provide reliable or reproducible extract products that are
not found in currently know extract products. For example, an
essential oil fraction or sub-fraction from one species may be
combined with an essential oil fraction or sub-fraction from the
same or different species or with a polyphenolic acid fraction from
the same or different species, and that combination may or may not
be combined with a polysaccharide fraction from the same or
different species of cinnamon.
[0147] Extractions of the disclosure may also be defined in terms
of concentrations relative to those found in natural cinnamon
species. Embodiments also comprise extractions wherein one or more
of the fractions, including essential oils, polyphenolic acids, or
polysaccharides, are found in a concentration that is greater than
that found in native cinnamon species plant material. Embodiments
also comprise extractions wherein one or more of the fractions,
including essential oils, polyphenolics, or polysaccharides, are
found in a concentration that is less than that found in native
cinnamon species. Known amounts of the bio-active chemical
constituent fractions of the cinnamon species (Table 1) are used as
an example of the disclosure. For example, extractions of the
disclosure comprise fractions wherein the concentration of
essential oils is from 0.001 to 50 times the concentration of
native cinnamon species, and/or compositions where the
concentration of desired polyphenolic acids is from 0.001 to 50
times the concentration of native cinnamon species, and/or
compositions where the concentration of water soluble-ethanol
insoluble polysaccharides is from 0.001 to 20 times the
concentration of native cinnamon species.
[0148] Extractions of the disclosure comprise fractions wherein the
concentration of essential oils is from 0.01 to 50 times the
concentration of native cinnamon species, and/or compositions
wherein the concentration of desired polyphenolic acids is from
0.01 to 50 times the concentration of native cinnamon species,
and/or compositions wherein the concentration of polysaccharides is
from 0.01 to 20 times the concentration of native cinnamon species.
Furthermore, extractions of the disclosure comprise sub-fractions
of the essential oil chemical constituents having at least one or
more of chemical compounds present in the native plant material
essential oil that is in amount greater or less than that found in
native cinnamon plant material essential oil chemical constituents.
For example, the chemical compound, trans-cinnamaldehyde, may have
it's concentration increased in an essential oil sub-fraction to
80% by % mass weight of the sub-fraction from its concentration of
60% by % mass weight of the total essential oil chemical
constituents in the native cinnamon plant material. In contrast,
trans-cinnamaldehyde may have it's concentration reduced in an
essential oil sub-fraction to about 6% by % mass weight of the
sub-fraction from it's concentration of about 60% by % mass weight
of the total essential oil chemical constituents in the native
plant material, a 10 fold decrease in concentration. Extractions of
the disclosure comprise fractions wherein the concentration of
specific chemical compounds in such novel essential oil
sub-fractions is either increase by about 1.1 to about 10 times or
decreased by about 0.1 to about 10 times that concentration found
in the native cinnamon essential oil chemical constituents.
[0149] Additional embodiments comprise extractions comprising
altered profiles (ratio distribution) of the chemical constituents
of the cinnamon species in relation to that found in the native
plant material or to currently available cinnamon species extract
products. For example, the essential oil fraction may be increased
or decreased in relation to the polyphenolic acids and/or
polysaccharide concentrations. Similarly, the polyphenolic acids or
polysaccharides may be increased or decreased in relation to the
other extract constituent fractions to permit novel constituent
chemical profile extractions for specific biological effects. By
combining the isolated and purified fractions of one or more of
essential oils, polyphenolics and/or polysaccharides, extractions
may be made that provide novel combinations of essential oils.
[0150] Methods of the disclosure comprise providing novel cinnamon
extractions for treatment and prevention of human disorders. For
example, a novel cinnamon species extraction for treatment of type
2 diabetes mellitus may have an increased polyphenolic fraction
concentration and reduced essential oil and polysaccharide fraction
concentrations, by % weight, than that found in the cinnamon
species native plant material or conventional known extraction
products. A novel cinnamon species extraction for anti-oxidant,
anti-blood vessel damage, and ischemic cerebrovascular disease may
have an increased essential oil and polyphenolic acid fraction and
a reduced polysaccharide fraction, by % weight, than that found in
the native cinnamon species plant material or conventional known
extraction products. Another example of a novel cinnamon species
extraction, for treatment of allergic disorders comprises a
fraction having an increased polyphenolic fraction concentration,
an increased polysaccharide fraction, and a reduced essential oil
fraction than that found in native cinnamon species plant material
or known conventional extraction products.
Methods of Extraction
[0151] The following methods as taught may be used individually or
in combination with the disclosed method or methods known to those
skilled in the art. The starting material for extraction is plant
material from one or more cinnamon species. The plant material may
be the any portion of the plant, though the bark is the most
preferred starting material.
[0152] The cinnamon species plant material may undergo
pre-extraction steps to render the material into any particular
form, and any form that is useful for extraction is contemplated by
the disclosure. Such pre-extraction steps include, but are not
limited to, that wherein the material is chopped, minced, shredded,
ground, pulverized, cut, or torn, and the starting material, prior
to pre-extraction steps, is dried or fresh plant material. A
preferred pre-extraction step comprises grinding and/or pulverizing
the cinnamon species bark material into a fine powder. The starting
material or material after the pre-extraction steps can be dried or
have moisture added to it. Once the cinnamon species plant material
is in a form for extraction, methods of extraction are contemplated
by the disclosure.
[0153] Methods of extraction of the disclosure comprise processes
disclosed herein. In general, methods of the disclosure comprise,
in part, methods wherein cinnamon species plant material is
extracted using supercritical fluid extraction (SFE) with carbon
dioxide as the solvent (SCCO.sub.2) that is followed by one or more
solvent extraction steps, such as, but not limited to, water,
hydroalcoholic, and affinity polymer absorbent extraction
processes. Additional other methods contemplated for the disclosure
comprise extraction of cinnamon species plant material using other
organic solvents, refrigerant chemicals, compressible gases,
sonification, pressure liquid extraction, high speed counter
current chromatography, molecular imprinted polymers, and other
known extraction methods. Such techniques are known to those
skilled in the art. In one aspect, extractions of the disclosure
may be prepared by a method comprising the steps depicted
schematically in FIGS. 1-5.
[0154] The disclosure includes processes for concentrating
(purifying) and profiling the essential oil and other lipid soluble
compounds from cinnamon plant material using SCCO.sub.2 technology.
The disclosure includes the fractionation of the lipid soluble
chemical constituents of cinnamon into, for example, an essential
oil fraction of high purity (high essential oil chemical
constituent concentration). Moreover, the disclosure includes a
SCCO.sub.2 process wherein the individual chemical constituents
within an extraction fraction may have their chemical constituent
ratios or profiles altered. For example, SCCO.sub.2 fractional
separation of the chemical constituents within an essential oil
fraction permits the preferential extraction of certain essential
oil compounds relative to the other essential oil compounds such
that an essential oil extract sub-fraction can be produced with a
concentration of certain compounds greater than the concentration
of other compounds. Extraction of the essential oil chemical
constituents of the cinnamon species with SCCO.sub.2 as taught in
the disclosure eliminates the use of toxic organic solvents and
provides simultaneous fractionation of the extracts. Carbon dioxide
is a natural and safe biological product and an ingredient in many
foods and beverages.
[0155] In performing the previously described extraction methods,
it was found that greater than 80% yield by mass weight of the
essential oil chemical constituents having greater than 95% purity
of the essential oil chemical constituents in the original dried
cinnamon bark feedstock of the cinnamon species can be extracted in
the essential oil SCCO.sub.2 extract fraction (Step 1A). Using the
methods as taught in Step 1B (SCCO.sub.2 Extraction and
Fractionation Processes), the essential oil yield was reduced due
to the fractionation of the essential oil chemical constituents
into highly purified (>90%) essential oil sub-fractions. In
addition, the SCCO.sub.2 extraction and fractionation process as
taught in this disclosure permits the ratios (profiles) of the
individual chemical compounds comprising the essential oil chemical
constituent fraction to be altered such that unique essential oil
sub-fraction profiles can be created for particular medicinal
purposes. For example, the concentration of the steroid essential
oil chemical constituents may be increased while simultaneous
reducing the concentration of the fatty acid compounds or visa
versa.
[0156] Using the methods as taught in Step 2 of this disclosure, a
water soluble fraction is achieved with a 4.8% mass weight yield
from the original cinnamon species feedstock having a 26.0%
concentration of total phenolic acids, a yield of about 10% mass
weight of the phenolic acid chemical constituents found in the
native cinnamon bark feedstock. However, this water solvent extract
does contain valuable water soluble-ethanol insoluble
polysaccharide chemical constituents. In addition, this extraction
step achieves about 100% yield of the water soluble, ethanol
insoluble polysaccharides found in the native cinnamon species
plant material. The polysaccharide concentration in this
water-soluble extraction fraction is about 27% by % dry mass weight
in this water soluble extract fraction. Using 95% ethanol to
precipitate the polysaccharides, a purified polysaccharide fraction
may be collected from this water leaching extract. The yield of the
polysaccharide fraction is about 1.3% by % mass weight based on the
cinnamon rhizome feedstock. Based on a colormetric analytical
method using dextran as reference standards, a purity of >95%
cinnamon polysaccharides compounds may be obtained.
[0157] Using the methods as taught in Step 3 of this disclosure, a
hydroalcoholic leaching fraction is achieved with a 17.6% yield
from the original cinnamon species feedstock having a 64%
concentration of phenolic acids, about 1/3 of the phenolic acids
being non-bioactive tannins. This further equates to about a 90%
yield of the phenolic acid related chemical constituents found in
the native cinnamon species plant material.
[0158] Using the methods as taught in Step 4 of this disclosure
(Affinity Adsorbent Extraction Processes or Process
Chromatography), polyphenolic acid fractions with purities of
greater than 95% by % dry mass of the extraction fraction with less
than 0.1% tannins by % mass weight may be obtained. It is possible
to extract about 77% of the non-tannin polyphenolic acids from the
hydroalcoholic leaching extract feedstock. This equates to a 69%
yield of the polyphenolic acid chemical constituents found in the
native cinnamon species plant material. Based on the average degree
of polymerization, the purified polyphenolic fractions are largely
made of the beneficial bioactive polyphenolic oligomers.
[0159] Furthermore, it is possible to profile the polyphenolic
chemical constituents of the purified polyphenolic fractions. For
example, purified polyphenolic sub-fractions may be obtained
containing a high concentration of polyphenolic trimers or
tetramers. Such novel purified polyphenolic sub-fractions may have
great value for specific medical conditions.
[0160] Finally, the methods as taught in the disclosure permit the
purification (concentration) of the cinnamon species essential oil
chemical constituent fractions, novel polyphenolic fractions or
sub-fractions, and a novel polysaccharide fraction to be as high as
99%% by mass weight of the desired chemical constituents in the
essential oil fractions, as high as 97% by mass weight in the
polyphenolic phenolic fraction, and as high as 98% by mass weight
in the polysaccharide fraction. The specific extraction
environments, rates of extraction, solvents, and extraction
technology used depend on the starting chemical constituent profile
of the source material and the level of purification desired in the
final extraction products. Specific methods as taught in the
disclosure can be readily determined by those skilled in the art
using no more than routine experimentation typical for adjusting a
process to account for sample variations in the attributes of
starting materials that is processed to an output material that has
specific attributes. For example, in a particular lot of cinnamon
species plant material, the initial concentrations of the essential
oil chemical constituents, the polyphenolic acids, and the
polysaccharides are determined using methods known to those skilled
in the art as taught in the disclosure. One skilled in the art can
determine the amount of change from the initial concentration of
the essential oil chemical constituents, for instance, to the
predetermined amounts or distribution (profile) of essential oil
chemical constituents for the final extraction product using the
extraction methods, as disclosed herein, to reach the desired
concentration and/or chemical profile in the final cinnamon species
extraction product.
[0161] A schematic diagram of the methods of extraction of the
biologically active chemical constituents of cinnamon is
illustrated in FIGS. 1-5. The extraction process is typically, but
not limited to, 4 steps.
Step 1: Supercritical Fluid Carbon Dioxide Extraction of Cinnamon
Essential Oil
[0162] Due to the hydrophobic nature of the essential oil,
non-polar solvents, including, but not limited to SCCO.sub.2,
hexane, petroleum ether, and ethyl acetate may be used for this
extraction process. Since some of the components of the essential
oil are volatile, steam distillation may also be used as an
extraction process.
[0163] A generalized description of the extraction of the essential
oil chemical constituents from the bark of the cinnamon species
using SCCO.sub.2 is diagrammed in FIG. 2-Step 2A and 2B. The
feedstock 10 is dried ground cinnamon bark (about 140 mesh). The
extraction solvent 210 is pure carbon dioxide. Ethanol may be used
as a co-solvent. The feedstock is loaded into a SFE extraction
vessel 20. After purge and leak testing, the process comprises
liquefied CO.sub.2 flowing from a storage vessel through a cooler
to a CO.sub.2 pump. The CO.sub.2 is compressed to the desired
pressure and flows through the feedstock in the extraction vessel
where the pressure and temperature are maintained at the desired
level. The pressures for extraction range from about 60 bar to 800
bar and the temperature ranges from about 35.degree. C. to about
90.degree. C. The SCCO.sub.2 extractions taught herein are
preferably performed at pressures of at least 100 bar and a
temperature of at least 35.degree. C., and more preferably at a
pressure of about 60 bar to 500 bar and at a temperature of about
40.degree. C. to about 80.degree. C. The time for extraction for a
single stage of extraction range from about 30 minutes to about 2.5
hours, to about 1 hour. The solvent to feed ratio is typically
about 60 to 1 for each of the SCCO.sub.2 extractions. The CO.sub.2
is recycled. The extracted, purified, and profiled essential oil
chemical constituents 30 are then collected a collector or
separator, saved in a light protective glass bottle, and stored in
a dark refrigerator at 4.degree. C. The cinnamon feedstock 10
material may be extracted in a one step process (FIG. 2, Step 2A)
wherein the resulting extracted and purified cinnamon essential oil
fraction 30 is collected in a one collector SFE or SCCO.sub.2
system 20 or in multiple stages (FIG. 2, Step 2B) wherein the
extracted purified and profiled cinnamon essential oil
sub-fractions 50, 60, 70, 80 are separately and sequentially
collected in a one collector SFE system 20. Alternatively, as in a
fractional SFE system, the SCCO.sub.2 extracted cinnamon feedstock
material may be segregated into collector vessels (separators) such
that within each collector there is a differing relative percentage
essential oil chemical constituent fraction (profile) in each of
the purified essential oil sub-fractions collected. The residue
(remainder) 40 is collected, saved and used for further processing
to obtain purified fractions of the cinnamon species phenolic acids
and polysaccharides. An embodiment of the disclosure comprises
extracting the cinnamon species feedstock material using
multi-stage SCCO.sub.2 extraction at a pressure of 60 bar to 500
bar and at a temperature between 35.degree. C. and 90.degree. C.
and collecting the extracted cinnamon material after each stage. A
second embodiment of the disclosure comprises extracting the
cinnamon species feedstock material using fractionation SCCO.sub.2
extraction at pressures of 60 bar to 500 bar and at a temperature
between 35.degree. C. and 90.degree. C. and collecting the
extracted cinnamon material in differing collector vessels at
predetermined conditions (pressure, temperature, and density) and
determined intervals (time). The resulting extracted cinnamon
purified essential oil sub-fractions from each of the multi-stage
extractors or in differing collector vessels (fractional system)
can be retrieved and used independently or can be combined to form
one or more cinnamon essential oil fractions comprising a
predetermined essential oil chemical constituent concentration that
is higher or lower than that found in the native plant material or
in conventional cinnamon extraction products. Typically, the total
yield of the essential oil fraction from cinnamon species using a
single step maximal SCCO.sub.2 extraction is about 0.4 to about
1.8% (>85% of the essential oil chemical constituents) by %
weight having an essential oil chemical constituent purity of
greater than 95% by mass weight of the extract. The results of such
extraction processes are found below and in Table 4. The procedure
can be found in Example 1. TABLE-US-00004 TABLE 4 HPLC analysis of
single stage SFE cinnamon essential oil extraction. Density CND CND
yield T (.degree. C.) P (bar) (g/cc) S/F Yield (%) purity (%) (%)
40 80 0.293 57 0.46 69.1 0.32 40 100 0.64 57 0.87 60.2 0.53 40 120
0.723 57 0.87 61.5 0.53 40 300 0.915 57 1.27 58.0 0.74 60 80 0.195
38 0.34 65.4 0.22 60 100 0.297 38 0.34 68.1 0.23 60 120 0.448 38
0.43 67.1 0.29 60 300 0.834 38 1.14 58.7 0.67 80 100 0.226 19 0.49
68.0 0.33 80 300 0.751 19 1.14 59.6 0.68
[0164] These results demonstrate the effect of pressure on the
kinetics of extraction. Higher extraction pressures result in the
system reaching equilibrium at shorter times with less amount of
CO.sub.2 consumed. The total extraction yield increases with
increasing extraction pressure due to the density increase
associated with pressure increase. Interestingly, a lower pressures
such as 100-300 bar, the lower the temperature, the higher the
yield again related to a higher density. At higher pressures such
as 300-500 bar, temperature has far less effect of the extraction
yield. Although a higher yield and greater efficiency of extraction
may be achieved with pressures greater than 200 bar, 95% purity of
the essential oil chemical constituents can be achieved with
pressures less than 300 bar and temperatures of about 40-80.degree.
C.
[0165] In the experiment range investigated, it can be clearly
noted that there is a competition effect between temperature and
density. This aspect is well defined and documented in the
literature, where an increase in pressure, at constant temperature,
leads to an increase in the yield due to the enhancement in the
solvency power of the supercritical and near critical fluid. An
increase in temperature promotes an enhancement in vapor pressure
of the compounds favoring the extraction. Additionally, the
increase in diffusion coefficient and the decrease in solvent
viscosity also help the compounds extraction from the herbaceous
porous matrix as the temperature is increased to higher value. On
the other hand, an increase in temperature, at constant system
pressure, leads to a decrease in the solvent density.
[0166] Seventy-one compounds were separated and identified in
cinnamon bark essential oil using GC-MS analysis. By comparing the
mass spectra data of sample with the data in the scientific
literature, cinnamaldehyde, coumarin, and cinnamyl acetate were
identified. (Tables 3 and 4) In addition to cinnamaldehyde and it's
cogeners such as benzaldhyde (P1), cinnamaldehyde (P10 & P14),
cinnamyl alcohol (P16), trans-cinnamic acid (P23), and cinnamyl
acetate (P25), 4 monoterpenes (P6, P8, and P9), 16 sesquiterpenes
(P20-22, P26, P29, P31-2, P35-42, and P46), and 9 fatty acids and
fatty acid derivatives were identified. Other minor aromatic and
aliphatic compounds were also present. Of the compounds identified,
SFE was able to extract fatty acids and steroid compounds that had
not previously been identified in cinnamon essential oil. These
compounds make up about 90% of the essential oil chemical
constituents by % mass weight. Cinnamaldehyde is the major chemical
constituent of the cinnamon essential oil at about 70-91% by % mass
weight. A greater number of compounds were identified from
extractions under the conditions of 40.degree. C. and 120 bar with
higher purity of about 100% than at SFE extraction conditions of
higher temperatures and pressures. Cinnamaldehyde purity of greater
than 90% mass weight was accomplished with SFE temperatures of
60.degree. C. and 100 bar with a loss of steroid compounds and
lower fatty acid and sesquiterpene purity. Steroid compounds can
only be extracted a low temperature of 40.degree. C. At a SFE
temperature of 40.degree. C. and 80 bar, the steroid compound
chemical constituent purity was as high as 20% mass weight. In
contrast, higher SFE temperatures (60-80.degree. C.) and pressures
(500 bar) favor the extraction of the fatty acid compounds. These
data indicate that SCCO.sub.2 has the ability to profile the
chemical constituents of cinnamon essential oil. TABLE-US-00005
TABLE 5 Compounds Identified in Cinnamon Essential Oil Fraction Ret
Peak time ID (min) Compound CAS# Formula Mw structure P1 7.2
Benzaldehyde 100-52-7 C7H6O 106 ##STR1## P2 9.9 Benzeneacetaldehyde
122-78-1 C8H8O 120 ##STR2## P3 10.6 Acetophenone 98-86-2 C8H8O 120
##STR3## P4 10.8 Benzoylcarboxaldehyde 1074-12-0 C8H6O2 134
##STR4## P5 14.1 Benzenepropanal 104-53-0 C9H10O 134 ##STR5## P6
14.3 Borneol 507-70-0 C10H18O 154 ##STR6## P7 14.6 Benzofuran,
2-methyl- 4265-25-2 C9H8O 132 ##STR7## P8 14.7 1-Terpinen-4-ol
562-74-3 C10H18O 154 ##STR8## P9 15.2 .alpha.-Terpieol 10482-56-1
C10H18O 154 ##STR9## P10 16.1 Cinnamylaldehyde 104-55-2 C9H8O 132
##STR10## P11 16.5 Benzenepropanol 122-97-4 C9H12O 136 ##STR11##
P12 16.8 Benzoylformic acid 611-73-4 C8H6O3 150 ##STR12## P13 17.5
Benzene, 1,3-bis(1,1- dimethylethyl)- 1014-60-4 C14H22 190
##STR13## P14 18.4 Cinnamaldehyde, (E)- 14371-10-9 C9H8O 132
##STR14## P15 18.8 Acetic acid, bornyl ester 92618-89-8 C12H20O2
196 ##STR15## P16 19.5 Cinnamyl alcohol 104-54-1 C9H10O 134
##STR16## P17 20.0 2,4-Decadienal 2363-88-4 C10H16O 152 ##STR17##
P18 20.5 2,4-dimethyl-1-heptanol 18450-74-3 C9H20O 144 ##STR18##
P19 22.0 Megastigam- 4,6(E),8(E)-triene 51468-86-1 C13H20 176
##STR19## P20 23.6 Copaene 3856-25-5 C15H24 204 ##STR20## P21 26.3
1,3,6,10- Dodecatetraene, 3,7,11-trimethyl-, (Z,E)- 26560-14-5
C15H24 204 ##STR21## P22 26.6 Beta-caryophyllene 87-44-5 C15H24 204
##STR22## P23 26.9 trans-Cinnamic acid 140-10-3 C9H8O2 148
##STR23## P24 27.4 Coumarin 91-64-5 C9H6O2 146 ##STR24## P25 28.5
Cinnamyl acetate 103-54-8 C11H12O2 176 ##STR25## P26 34.0
.alpha.-Muurolene 31983-22-9 C15H24 204 ##STR26## P27 34.2
3-(phenylmethoxy)-1- propanol 4799-68-2 C10H14O2 166 ##STR27## P28
35.0 Phenol, 3,5-bis(1,1- dimethylethyl)- 1138-52-9 C14H22O 206
##STR28## P29 35.7 (-)-Calamenene 483-77-2 C15H22 202 ##STR29## P30
35.9 Cinnamaldehyde, o- methoxy- 1504-74-1 C10H10O2 162 ##STR30##
P31 36.4 1,2,3,4,4A,7- hexahydro-1,6- dimethyl-4-(1- methylethyl)-
naphthalene 16728-99-7 C15H24 204 ##STR31## P32 39.3
.beta.-Caryophyllene epoxide 1139-30-6 C15H24O 220 ##STR32## P33
40.7 unknown 1 P34 41.5 Benzaldehyde, 4- propyl- 28785-06-0 C10H12O
148 ##STR33## P35 41.8 Cubenol 21284-22-0 C15H26O 222 ##STR34## P36
42.1 .alpha.-Cadinol 481-34-5 C15H26O 222 ##STR35## P37 42.4
delta-cardinol 36563-42-8 C15H26O 222 ##STR36## P38 42.6
.alpha.-muurolol 19435-97-3 C15H26O 222 ##STR37## P39 42.9
.tau.-muurolol 19912-62-0 C15H26O 222 ##STR38## P40 43.1 Germacrene
D 23986-74-5 C15H24 204 ##STR39## P41 43.3 .alpha.-Cubebene
17699-14-8 C15H24 204 ##STR40## P42 43.7 1H- Cycloprop[e]azulene,
decahydro-1,1,4,7- tetramethyl-,[1aR- (1a.alpha.,4.beta.,4a.be-
ta.,7.beta.,7a.beta.,7b.al- pha.)]- 28580-43-0 C15H26 206 ##STR41##
P43 43.8 Naphthalene, 1,6- dimethyl-4-(1- methylethyl)- 483-78-3
C15H18 198 ##STR42## P44 unknown P45 44.7 2-Propenoic acid,
tridecyl ester 4/8/3076 C16H30O2 254 ##STR43## P46 47.6
1,2,3,4,4A,7- hexahydro-1,6- dimethyl-4-(1- methylethyl)-
naphthalene 16728-99-7 C15H24 204 ##STR44## P47 48.6 Propanoic
acid, 3- hydroxy-3-phenyl-,t- butyl ester 5397-27-3 C13H18O3 222
##STR45## P48 49.7 2-Dodecanol, 2-methyl- 1653-37-8 C13H28O 200
##STR46## P49 51.1 1-Hexadecanol 36653-82-4 C16H34O 242 ##STR47##
P50 52.4 pentadecanoic acid, methyl ester 7132-64-1 C16H32O2 256
##STR48## P51 52.6 1,19-Eicosadiene 14811-95-1 C20H38 278 ##STR49##
P52 53.4 n-Hexadecanoic acid 57-10-3 C16H32O2 256 ##STR50## P53
56.0 Oleyl Alcohol 143-28-2 C18H36O 268 ##STR51## P54 56.6
1-Nonadecanol 1454-84-8 C19H40O 284 ##STR52## P55 57.9 Ethanol,
2-(9,12- octadecadienyloxy)-, (Z,Z)- 17367-08-7 C20H38O2 310
##STR53## P56 58.0 9-Octadecenoic acid (Z)- 112-80-1 C18H34O2 282
##STR54## P57 58.2 unknowns unknowns P58 58.6 Eicosanoic acid
506-30-9 C20H40O2 312 ##STR55## P59 59.1 Hexadecanoic acid, butyl
ester 111-06-8 C20H40O2 312 ##STR56## P60 63.8 Octadecanoic acid,
butyl ester 123-95-5 C22H44O2 340 ##STR57## P61 64.1 Heneicosane
629-94-7 C12H44 296 ##STR58## P62 64.9 Benzenepropanoic acid, 10-
oxotricyclo[4.2.1.1(2,5)]deca-3,7-dienyl ester 0-00-0 C19H18O3 294
P63 66.0 Cyclopentanemethanol, 2-nitro-.alpha.-(2- phenylethenyl)-,
[1.alpha.(S@),2.alpha.]- 103130-01-4 C14H17NO3 247 ##STR59## P64
7,22-Ergostadienol C28H46O 398 ##STR60## P65 67.7 unknown P66 68.6
1,2- Benzenedicarboxylic acid, diisooctyl ester 27554-26-3 C24H38O4
390 ##STR61## P67 68.7 .beta.-Sitosterol 83-46-5 C29H50O 414
##STR62## P68 70.6 Ergosta-7,22-dien-3-ol, (3.beta.,22E)-
17608-76-3 C28H46O 398 ##STR63## P69 72.6 4,4,6a,6b,8a,11,11,14b-
Octamethyl- 1,4,4a,5,6,6a,6b,7,8,8a, 9,10,11,12,12a,14,14a,
14b-octadecahydro-2H- picen-3-one C30H48O 424 ##STR64## P70 74.4
Ergosta-7,22-dien-3-ol, (3.beta.,5. alpha., 22E)- 11/4/2645 C28H46O
398 ##STR65## P71 76.9 Chondrillasterol 481-17-4 C29H48O 412
##STR66##
[0167] TABLE-US-00006 TABLE 6 GC-MS analysis peak area, peak area
percentage and calculated weight percentage of cinnamon bark
essential oil extracted at different conditions. T = 40.degree. C.,
P = 300 bar T = 80.degree. C., P = 100 bar T = 80.degree. C., P =
300 bar Ret. peak peak peak Peak time area area area No. (min) peak
area % Weight % peak area % Weight % peak area % Weight % P1 7.201
107275 0.03 0.03 3572872 0.57 0.57 161232 0.04 0.04 P2 9.866 239555
0.04 0.04 P3 10.649 94664 0.02 0.02 P4 10.822 275862 0.04 0.04 P5
14.054 29489 0.01 0.01 160992 0.04 0.04 P6 14.282 358823 0.11 0.11
386330 0.09 0.09 P7 14.563 374620 0.12 0.12 432491 0.1 0.1 P8
14.692 400042 0.12 0.12 413562 0.1 0.1 P9 15.153 683952 0.21 0.21
200566 0.03 0.03 890990 0.21 0.21 P10 16.112 1672873 0.51 0.51
4210512 0.67 0.67 1090882 0.26 0.26 P11 16.528 98413 0.03 0.03
305036 0.05 0.05 264483 0.06 0.06 P12 16.848 138317 0.02 0.02 P13
17.471 314610 0.1 0.1 451375 0.07 0.07 244719 0.06 0.06 P14 18.388
228756437 70.29 70.29 560967679 89.76 89.76 358267571 86.02 86.02
P15 18.798 804095 0.25 0.25 680662 0.16 0.16 P16 19.499 1329676
0.41 0.41 3744998 0.6 0.6 3918089 0.94 0.94 P17 20.038 189718 0.06
0.06 215682 0.03 0.03 156726 0.04 0.04 P18 20.494 86462 0.03 0.03
60887 0.01 0.01 P19 21.954 176284 0.05 0.05 165025 0.03 0.03 205784
0.05 0.05 P20 23.566 2473168 0.76 0.76 1468434 0.35 0.35 P21 26.287
137651 0.04 0.04 206073 0.05 0.05 P22 26.592 367241 0.11 0.11
802082 0.19 0.19 P23 26.908 339296 0.08 0.08 P24 27.432 15068316
4.63 4.63 23526861 3.77 3.77 25045782 6.01 6.01 P25 28.466 3071028
0.94 0.94 12812414 2.05 2.05 4063398 0.98 0.98 P26 33.953 387927
0.12 0.12 281914 0.05 0.05 553811 0.13 0.13 P27 34.237 281619 0.05
0.05 P28 35.035 111608 0.03 0.03 85138 0.02 0.02 P29 35.674 158637
0.05 0.05 551592 0.09 0.09 290965 0.07 0.07 P30 35.881 119357 0.04
0.04 422786 0.07 0.07 486137 0.12 0.12 P31 36.356 72181 0.02 0.02
118059 0.03 0.03 P32 39.334 335593 0.1 0.1 1272069 0.2 0.2 460812
0.11 0.11 P33 40.650 49352 0.02 0.02 P34 41.464 83100 0.03 0.03
246325 0.06 0.06 P35 41.750 176194 0.05 0.05 924151 0.15 0.15
354941 0.09 0.09 P36 42.087 181715 0.06 0.06 160919 0.03 0.03
245345 0.06 0.06 P37 42.390 93210 0.03 0.03 629354 0.1 0.1 P38
42.586 311094 0.05 0.05 86504 0.02 0.02 P39 42.927 331623 0.05 0.05
117555 0.03 0.03 P40 43.140 165961 0.05 0.05 795638 0.13 0.13
469680 0.11 0.11 P41 43.339 279943 0.04 0.04 154116 0.04 0.04 P42
43.672 144713 0.02 0.02 85123 0.02 0.02 P43 43.789 125052 0.02 0.02
P44 78201 0.02 0.02 P45 44.739 153149 0.05 0.05 861436 0.14 0.14
205244 0.05 0.05 P46 47.604 232837 0.04 0.04 P47 48.648 86340 0.03
0.03 201161 0.03 0.03 158427 0.04 0.04 P48 49.657 100070 0.03 0.03
104638 0.03 0.03 P49 51.066 104471 0.03 0.03 819892 0.13 0.13
494453 0.12 0.12 P50 52.420 106293 0.02 0.02 P51 52.609 102916 0.02
0.02 P52 53.430 132985 0.04 0.04 930693 0.15 0.15 1910179 0.46 0.46
P53 55.979 460792 0.07 0.07 455416 0.11 0.11 P54 56.611 287130 0.05
0.05 360772 0.09 0.09 P55 57.895 1058073 0.25 0.25 P56 57.997
1451917 0.35 0.35 P57 58.195 189737 0.06 0.06 123292 0.02 0.02
1178268 0.28 0.28 P58 58.550 678103 0.16 0.16 P59 59.112 932215
0.29 0.29 850903 0.14 0.14 999986 0.24 0.24 P60 63.761 1876786 0.58
0.58 1438157 0.35 0.35 P61 64.109 146342 0.04 0.04 P62 64.927
327089 0.08 0.08 P63 66.005 619225 0.15 0.15 P64 67.234 9979848
3.07 3.07 P65 67.746 68066 0.02 0.02 P66 68.642 152882 0.04 0.04
P67 68.651 6629886 2.04 2.04 P68 70.645 16769518 5.15 5.15 P69
72.590 10386507 3.19 3.19 P70 74.399 10804061 3.32 3.32 P71 76.879
686165 2.67 2.67 total 325266746.0 100.0 100.0 622411230.0 99.6
99.6 414900414.0 99.6 99.6 cinna- 234937289.0 72.2 72.2 585308475.0
93.7 93.7 367840468.0 88.3 88.3 maldehyde congeners aromaric
251223142.0 77.2 77.2 611370763.0 97.8 97.8 395724862.0 95.0 95.0
compounds nomoterpene 1442817.0 0.4 0.4 200566.0 0.0 0.0 1690882.0
0.4 0.4 sesquiterpene 4390841.0 1.3 1.3 5364255.0 0.9 0.9 5122535.0
1.2 1.2 fatty acid and 3489413.0 1.1 1.1 4543347.0 0.7 0.7
10481548.0 2.5 2.5 its derivatives steroids 63255985.0 19.4 19.4
0.0 0.0 0.0 0.0 0.0 0.0 Note: weight % were calculated by: Weight %
= (weight of each compound/total weight of extracts) .times. 100
where weight of each compound = peak area percentage .times. total
weight of extracts.
Step 2. Water Leaching Process and Polysaccharide Precipitation
[0168] The polysaccharide extract fraction of the chemical
constituents of cinnamon species has been defined in the scientific
literature as the "water soluble, ethanol insoluble extraction
fraction". A generalized description of the extraction of the
polysaccharide fraction from extracts of cinnamon species using
water solvent leaching and ethanol precipitation processes is
diagrammed in FIG. 3-Step 2. The feedstock 10 or 40 is native
ground cinnamon species plant material or the solid residue from
the SFE extraction process of Step 1. This feedstock is leaching
extracted in two stages. The solvent is distilled water 220. In
this method, the cinnamon species feedstock 10 or 40 and the
extraction solvent 220 are loaded into an extraction vessel 100,
110 and heated and stirred. It may be heated to 100.degree. C., to
about 80.degree. C., or to about 80-90.degree. C. The extraction is
carried out for about 1-5 hours, for about 2-4 hours, or for about
2 hours. The two stage extraction solutions 300+320 are combined
and the slurry is filtered 120, centrifuged 130, and the
supernatant collected and evaporated 140 to remove water until an
about 8-fold increase in concentration of the chemicals in solution
330. Anhydrous ethanol 230 is then used to reconstitute the
original volume of solution making the final ethanol concentration
at 95%. A large precipitate 150 is observed. The solution is
centrifuged 160, decanted 170 and the supernatant residue 340 may
be saved for further processing. The precipitate product 350 is the
purified polysaccharide fraction that may be analyzed for
polysaccharides using the colormetric method by using Dextran
5,000-410,000 molecular weight as reference standards. The actual
procedure can be found in Example 3. The purity of the extracted
polysaccharide fraction using 3 different molecular weight dextran
as standards is about 29, 35, and 47%, respectively, with a total
yield of 1.3% by % mass weight of the original native cinnamon bark
feedstock. Combining the purity measures of the 3 dextran standards
indicates a very high level of purity of greater than 95%.
Moreover, AccuTOF-DART mass spectrometry (see Exemplification
section) was used to further profile the molecular weights of the
compounds comprising the purified polysaccharide fraction. The
actual procedure can found in the Exemplification section.
Step 3. Hydroalcoholic Leaching Process for Extraction of Crude
Polyphenolic Acid Fraction
[0169] In one aspect, the disclosure comprises extraction and
concentration of the bio-active polyphenolic acid chemical
constituents. A generalized description of this step is diagrammed
in FIG. 4-STEP 3. This Step 2 extraction process is a solvent
leaching process. The feedstock for this extraction is either
cinnamon species ground dry bark material 10 or the residue 40 or
330+340 from the Step 1 SCCO.sub.2 extraction of the essential oil
chemical constituents or the Step 2 polysaccharide
extraction-precipitation, respectively. The extraction solvent 240
is aqueous ethanol. The extraction solvent may be 10-95% aqueous
alcohol, 25% aqueous ethanol is preferred. In this method, the
cinnamon feedstock material and the extraction solvent are loaded
into an extraction vessel 400 that is heated and stirred. It may be
heated to 100.degree. C., to about 90.degree. C., to about
80.degree. C., to about 70.degree. C., to about 60.degree. C. or to
about 30-50.degree. C. The extraction is carried out for about 1-10
hours, for about 1-5 hours, for about 2 hours. The resultant
extract solution is filtered 410 and centrifuged 420. The filtrate
(supernatant) 500, 520, 540 is collected as product, measured for
volume and solid content dry mass after evaporation of the solvent.
The extraction residue material 530 may be retained and saved for
further processing or discarded. The extraction may be repeated as
many times as is necessary or desired. It may be repeated 2 or more
times, 3 or more times, 4 or more times, etc. For example, FIG.
1-STEP 2 shows a three stage process, where the second stage and
the third stage use the same methods and conditions
[0170] Interestingly, residual cinnamaldehyde was extracted with
this hydroalcoholic leaching extraction process indicating that not
all of the essential oil chemical constituents were extracted with
relatively exhaustive extraction using the above SFE conditions.
Moreover, a significant amount of tannins were extracted making up
greater than 20% of the extraction product. Moreover, a two stage
hydroalcoholic leaching process is preferred to achieve a high
extraction yield of polyphenolics (about 18% by mass weight based
on the raw feedstock material) with a total phenolic acid
concentration of about 64% by mass weight and a tannin acid
concentration of about 20% by mass weight. In order to develop a
purified polyphenolic fraction containing a high concentration of
bioactive polyphenolics, an additional processing step (Step 4) is
required to remove the tannins from the crude Step 3 polyphenolic
fraction.
Step 4. Affinity Adsorbent Polyphenolic Extraction and Purification
Process
[0171] The beneficial bioactive polyphenolic acids are
proanthocyanidins. Proanthocyanidin are known as condensed tannins.
They are ubiquitous and present as the second most abundant natural
plant polyphenolics after lignins. Dubois M et al. Analytical Chem
28:350-356, 1956. The proanthocyanidins are mixtures of oligomers
and polymers consisting of (+)-catechin and/or (-)-epicatechin
units linked mainly through C4-C8 and/or C4-C6 bonds (B-type).
These flavan-3-ol can be double linked by a C4-C8 bond and an
additional ether bond between O7-C2 (A type). The molecular weight
of proanthocyanidins expressed as degree of polymerization (DPn) is
one of the most important properties. As defined in the scientific
literature, DP1 is a monomer, DP2-10 are oligomers, and DP>10
are polymers, respectively.
[0172] In the biomedical literature regarding cinnamon
polyphenolics (see above), DP 4-5 (oligomers) exhibit the medically
beneficial biological activity. Therefore, in Step 4 processing,
tannin removal and proanthocyanidin extraction and purification has
been studied by tracking total phenolic acid concentration and DPn
in each step of processing.
[0173] As taught herein, a purified polyphenolic acid fraction
extract from cinnamon and related species may be obtained by
contacting a hydroalcoholic extract of cinnamon feedstock with a
solid affinity polymer adsorbent resin so as to adsorb the
polyphenolic acids contained in the hydro-alcoholic extract onto
the affinity adsorbent. The bound chemical constituents are
subsequently eluted by the methods taught herein. Prior to eluting
the polyphenolic acid fraction chemical constituents, the affinity
adsorbent with the desired chemical constituents adsorbed thereon
may be separated from the remainder of the extract in any
convenient manner, preferably, the process of contacting with the
adsorbent and the separation is effected by passing the aqueous
extract through an extraction column or bed of the adsorbent
material.
[0174] A variety of affinity adsorbents can be utilized to purify
the phenolic acid chemical constituents of cinnamon species, such
as, but not limited to Sephadex LH-20 (Sigma Aldrich Co.),
"Amberlite XAD-2" (Rohm & Hass), "Duolite S-30" (Diamond Alkai
Co.), "SP207" (Mitsubishi Chemical), ADS-5 (Nankai University,
Tianjin, China), ADS-17 (Nankai University, Tianjin, China), Dialon
HP 20 (Mitsubishi, Japan), and Amberlite XAD7 HP (Rohm &
Hass).
[0175] Sephadex LH020 is preferably used for process chromatography
due to the high affinity for the polyphenolic acid chemical
constituents of and its ability to separate tannin polyphenolics
from non-tannin polyphenolics. The tannin polyphenolics adsorb to
Sephadex LH-20 in alcohol. In contrast non-tannin polyphenoics can
be eluted from the resin beads using alcohol whereas the tannins
remain adsorb on the beads. The tannins can then be eluted later
with aqueous acetone. This method permits the separation of the
tannin polyphenolic from the desired non-tannin polyphenolics of
cinnamon. Thus, different elution solvents can be used for the
separation of the polyphenolic compounds and purification of the
non-tannin bioactive cinnamon polyphenolics. Using the
Folin-Ciocalteu method and the protein-precipitable phenolic
method, the tannin and non-tannin polyphenolic concentrations can
be measured in the crude extraction fraction and the elution
fractions.
[0176] Although various eluants may be employed to recover the
non-tannin polyphenolic acid chemical constituents from the
adsorbent, in one aspect of the disclosure, the eluant comprises
low molecular weight alcohols, including, but not limited to,
methanol, ethanol, or propanol. In a second aspect, the eluant
comprises low molecular alcohol in an admixture with water. In
another aspect, the eluant comprises low molecular weight alcohol,
a second organic solvent, and water.
[0177] Although various eluants may be employed to recover the
tannin polyphenolic acid chemical constituents, in one aspect of
the disclosure, the eluant comprises aqueous acetone.
[0178] Preferably, the cinnamon species feedstock has undergone a
one or more preliminary purification process such as, but not
limited to, the processes described in Step 1 and 3 prior to
contacting the aqueous phenolic acid chemical constituent
containing extract with the affinity adsorbent material.
[0179] Using affinity adsorbents as taught in the disclosure
results in highly purified bioactive polyphenolic oligomers
(DP2-10) acid chemical constituents of the cinnamon species that
are remarkably free of other chemical constituents which are
normally present in natural plant material or in available
commercial extraction products. For example, the processes taught
in the disclosure can result in purified polyphenolic acid extracts
that contain total phenolic acid chemical constituents in excess of
95% by dry mass weight containing only trace tannin
polyphenolics.
[0180] The extraction and purification of the bioactive
polyphenolic acids from the bark of the cinnamon species using
polymer affinity adsorbent resin beads is diagrammed in FIG. 1-Step
4. The feedstock for this extraction process may be the aqueous
ethanol solution containing the phenolic acids from Step 3
hydroalcoholic Leaching Extraction 500+/-520+/-540. The appropriate
weight of adsorbent resin beads (22 mg of polyphenolic acids per gm
of adsorbent resin) is washed (soaked) with 4-5 BV of 95% ethanol
250 prior to being packed into a column 620. The polyphenolic acid
containing aqueous solution 500+520 is concentrated using
evaporation to 1% of its original volume. Then, absolute ethanol
260 is added to the concentrated sample sufficient to increase the
volume 20 times, dissolving the polyphenolics in a 95% ethanol
solution. This solution is centrifuged 640 to remove any insoluble
material and the supernatant collected as the loading sample 550.
The loading sample 550 is loaded onto the column 650. Once the
column is fully loaded, the column is eluted 660 with 95% ethanol
270 at a flow rate of 2-3 BV/hour to elute the bioactive non-tannin
polyphenolics in an isocratic fashion from the affinity adsorbent
column. The eluant 700 is collected in 1 BV fractions. The
polyphenolic fractions are each tested by UV spectrophotometer at
280 nm (polyphenolic acid wave length absorbance) until the
absorbance is not longer detected in the fraction samples at which
time the elution is discontinued. Generally 7-10 BV of 95% ethanol
are required to elute the non-tannin polyphenolics from the column
(about 3-4 hours). The eluted column 670 is washed 680 with 3 BV of
70% aqueous acetone 280 eluting the tannin polyphenolics adsorbed
on the resin beads at a flow rate of 5 BV/hr (3 hours). The eluted
tannin polyphenolic washing 710 is discarded 730. The washed column
730 is then washed with 4-5 95% ethanol 250 at a flow rate of 5
BV/hr to remove any remaining chemicals in the column preparing the
washed column for further process chromatography 740. The washing
720 is discarded 730. The elution fraction volumes 700 may be
collected about every 1 BV and these samples are analyzed total
polyphenolics (Folin-Ciocalteu method), tannin polyphenolics
(Protein-precipitation Method, DPn (Thiolytic degradation HPLC) and
tested for solids content and purity.
[0181] The oligomeric and polymeric proanthocyanidin polyphenolic
compounds are eluted on a wide retention window (retention times
12-30 min) causing baseline deviation and difficulty with precise
integration of the chromatographic peaks when calculating the
catechin and epicatechin concentration. This HPLC behavior has been
verified for most proanthocyanidins in the scientific literature.
However, after thiolysis, the HPLC chromatograms clearly show
evidence of the improvement of chromatographic resolution. With
tholysis, the proanthocyanidins are converted into monomeric units
yielding well-resolved peaks on the HPLC chromatograms.
Benzylthioethers result from the extension unit of proanthocyanidin
structures according to the scientific literature (see Guyot 2001).
The DPn can be calculated by the total area of P1, P2, P3, and P4
and the total area of catechin and epicatechin.
[0182] Sephadex LH-20 has been shown to be an efficient affinity
adsorbent for the separation of tannin from nontannin polyphenolic
compounds in cinnamon hydroalcoholic extracts. Combining elution
fractions F2-F8 about 77.4% the non-tannin polyphenolic chemical
constituents can be recovered with only 0.2% of the tannins being
recovered in this combined extraction fraction. The yield of
combining elution fractions F2-F8 is 21.5% by mass weight of the
loading solution and 3.78% by mass weight based on the raw cinnamon
feedstock. The non-tannin polyphenolic purity is 65% by mass dry
weight which is 3 times higher than the crude polyphenolic
extraction product of Step 3. Moreover, a purity of greater than
95% by % mass weight can be found by combining elution fractions
F6-F8.
[0183] The average degree of polymerization (DPn) demonstrates the
size of the polyphenolic oligomer in each elution fraction. In the
crude extract (loading solution), the degree of polymerization was
6.9 due to the presence of the large tannin polyphenolic polymers.
In the polyphenolic elution fractions, essentially no tannin
polyphenolics were found. Therefore, the purified polyphenolic
elution fractions are made up largely of polyphenolic oligomers, a
mixture of dimers-DPn=2; trimers-DPn=3; tetamers-DPn=4; etc.). As
shown in Table 5, more trimers were eluted in elution fractions
F3-F5 and more tetramers were eluted in elution fractions F6-F8.
The range of DPn in the elution fractions was from 2.7 to 4.2
confirming that these fractions contain a high level purity of the
beneficial bioactive proanthocyanidin polyphenolic chemical
constituents of cinnamon. Furthermore, by combining different
elution fractions, different extraction products having different
purities of the nontannin polyphenolic and yields can be achieved
as demonstrated in Tables 7 and 8. TABLE-US-00007 TABLE 7 Analysis
of 95% ethanol elutions of polyphenolic fractions from Sephadex
LH-20 process chromatography. Weight (mg) Purity (%) Total Non
Yield Total phenolic Tannin Nontannin Tannin tannin Average Name
(%) solid acid acid acid acid acid DPn Loading 132.1 61.2 32.8 28.5
21.6 24.8 6.9 Elution F2 37.1 49.0 3.7 0.1 3.6 7.1 0.1 3.6 Elution
F3 7.4 9.8 2.9 0.0 2.9 29.5 0.0 2.7 Elution F4 5.2 6.8 4.5 0.0 4.5
66.4 0.0 3.6 Elution F5 3.2 4.2 3.7 0.0 3.7 87.8 0.0 3.1 Elution F6
2.3 3.1 2.9 0.0 2.9 91.1 0.0 4.0 Elution F7 2.1 2.8 2.9 0.0 2.9
100.0 0.0 4.2 Elution F8 1.2 1.6 1.6 0.0 1.6 93.8 0.0 4.2 Combine
5.7 7.5 7.3 0.0 7.3 97.2 0.0 4.1 .perp. 0.1 F6-F8 Combine 21.5 28.4
18.5 0.0 18.5 65.1 0.0 3.6 .perp. 0.6 F2-F8 Recovery 58.5 36.1 0.2
77.4 (%) * Elution 1 was not tabulated because there was chemical
constituents, only solvent.
[0184] TABLE-US-00008 TABLE 8 Results of yield and purity of
different nontannin polyphenolic elution fractions. Total Total
Total phenolic solid phenolic acid purity Yield based on Fractions
(mg) acid (mg)* (%) feedstock (%) DPn F2-F7 28.4 18.5 65.1 3.8 3.6
F3-F7 18.6 15.6 83.7 2.5 3.8 F4-F7 11.8 11.0 93.8 1.6 3.9 F5-F7 7.5
7.3 97.2 1.0 4.1 F6-F7 4.4 4.4 100.0 0.6 4.2 *Total phenolic acid
have no measurable tannin acids in these combined fractions.
[0185] Many methods are known in the art for removal of alcohol
from solution. If it is desired to keep the alcohol for recycling,
the alcohol can be removed from the solutions, after extraction, by
distillation under normal or reduced atmospheric pressures. The
alcohol can be reused. Furthermore, there are also many methods
known in the art for removal of water from solutions, either
aqueous solutions or solutions from which alcohol was removed. Such
methods include, but not limited to, spray drying the aqueous
solutions onto a suitable carrier such as, but not limited to,
magnesium carbonate or maltodextrin, or alternatively, the liquid
can be taken to dryness by freeze drying or refractive window
drying.
Food and Medicaments
[0186] As a form of foods of the present invention, there may be
formulated to any optional forms, for example, a granule state, a
grain state, a paste state, a gel state, a solid state, or a liquid
state. In these forms, various kinds of substances conventionally
known for those skilled in the art which have been allowed to add
to foods, for example, a binder, a disintegrant, a thickener, a
dispersant, a reabsorption promoting agent, a tasting agent, a
buffer, a surfactant, a dissolution aid, a preservative, an
emulsifier, an isotonicity agent, a stabilizer or a pH controller,
etc. may be optionally contained. An amount of the elderberry
extract to be added to foods is not specifically limited, and for
example, it may be about 10 mg to 5 g, preferably 50 mg to 2 g per
day as an amount of take-in by an adult weighing about 60 kg.
[0187] In particular, when it is utilized as foods for preservation
of health, functional foods, etc., it is preferred to contain the
effective ingredient of the present invention in such an amount
that the predetermined effects of the present invention are shown
sufficiently.
[0188] The medicaments of the present invention can be optionally
prepared according to the conventionally known methods, for
example, as a solid agent such as a tablet, a granule, powder, a
capsule, etc., or as a liquid agent such as an injection, etc. To
these medicaments, there may be formulated any materials generally
used, for example, such as a binder, a disintegrant, a thickener, a
dispersant, a reabsorption promoting agent, a tasting agent, a
buffer, a surfactant, a dissolution aid, a preservative, an
emulsifier, an isotonicity agent, a stabilizer or a pH
controller.
[0189] An administration amount of the effective ingredient
(cinnamon extract) in the medicaments may vary depending on a kind,
an agent form, an age, a body weight or a symptom to be applied of
a patient, and the like, for example, when it is administrated
orally, it is administered one or several times per day for an
adult weighing about 60 kg, and administered in an amount of about
10 mg to 5 g, preferably about 50 mg to 2 g per day. The effective
ingredient may be one or several components of the cinnamon
extract.
[0190] Methods also comprise administering such extracts more than
one time per day, more than two times per day, more than three
times per day and in a range from 1 to 15 times per day. Such
administration may be continuously, as in every day for a period of
days, weeks, months, or years, or may occur at specific times to
treat or prevent specific conditions. For example, a person may be
administered cinnamon species extracts at least once a day for
years to enhance mental focus, cognition, and memory, or to prevent
and treat type 2 diabetes mellitus, to prevent cardiovascular
disease stroke, or to treat gastro-intestinal disorders, or to
treat inflammatory disorders and arthritis including gout, or to
treat the common cold, bacterial and fungal infections.
[0191] The foregoing description includes the best presently
contemplated mode of carrying out the disclosure. This description
is made for the purpose of illustrating the general principles of
the disclosures and should not be taken in a limiting sense. This
disclosure is further illustrated by the following examples, which
are not to be construed in any way as imposing limitations upon the
scope thereof. On the contrary, it is to be clearly understood that
resort may be had to various other embodiments, modifications, and
equivalents thereof, which, after reading the description herein,
may suggest themselves to those skilled in the art without
departing from the spirit of the disclosure.
[0192] All terms used herein are considered to be interpreted in
their normally accepted usage by those skilled in the art. Patent
and patent applications or references cited herein are all
incorporated by reference in their entireties.
EXEMPLIFICATION
Materials
[0193] Acetone (67-64-1), >99.5%, ACS reagent (179124);
Acetonitrile (75-05-8), for HPLC, gradient grade .gtoreq.99.9% (GC)
(000687); Hexane (110-54-3), 95+%, spectrophotometric grade
(248878); Ethyl acetate (141-78-6), 99.5+%, ACS grade (319902);
Ethanol, denatured with 4.8% isopropanol (02853); Ethanol
(64-17-5), absolute, (02883); Methanol (67-56-1), 99.93%, ACS HPLC
grade, (4391993); and Water (7732-18-5), HPLC grade, (95304). All
were purchased from Sigma-Aldrich.
[0194] Formic acid (64-18-6), 50% solution (09676); Acetic acid
(64-19-7), 99.7+%, ACS reagent (320099); Hydrochloric acid
(7647-01-0), volumetric standard 1.0N solution in water (318949);
Calcium hydroxide (7789-78-8), powder, CA 0-2 mm, 90-95% (213268);
Ferric chloride anhydrous (7705-08-0), 97%, reagent grade(157740);
Folin-Clocalteu phenol reagent (2N) (47641); Phenol (108-95-2)
(P3653); Sulfuric acid (7664-93-9), ACS reagent, 95-97% (44719);
Triethanolamine(102-71-6), triethanolamine free base (T1377);
Sodium dodecyl sulfate(151-21-3), minimum 98.5% GC (L4509); all
were purchased from Sigma-Aldrich. Sodium carbonate (S263-1, Lot #:
037406) was purchased from Fisher Co.
[0195] Serum albumin (9048-46-8), Albumin Bovine Fraction V powder
cell culture tested (A9418); (+)-catechin hydrate (88191-48-4),
purity >98% (C1251); Gallic acid (149-91-7), ACS reagent,
.gtoreq.98% (HPLC); Benzylthiol (100-53-8), 99% (B25401);
Trans-cinnamaldehyde (14371-10-9), 99+% purity; tannin acid
(1401-55-4), powder (T0125); all were purchased from Sigma-Adrich.
(-)-epicatechin 93.6% (05125-550, CAS# 490-46-0) was purchased from
Chromadex. Dextran standard 5000 (00269), 50,000 (00891) and
410,000 (00895) certified according to DIN were purchased from
Fluka. The structures of chemical reference standards used in the
disclosure are shown below: ##STR67##
[0196] Sephadex LH-20: Sephadex.TM. LH-20 (Lot #: 308822, pack
167600, product #: 17-0090-01) were purchased from Ambersham
Bioscience AB Uppsala Sweden. It is prepared by hydroxypropylation
of sephadex G-25, a bead-formed dextran medium, and has been
specifically developed for gel filtration of natural products, such
as steroids, terpenoids, lipids and low molecular weight peptides,
in organic solvent.
HPLC Method
[0197] Chromatographic system: Shimadzu high Performance Liquid
Chromatographic LC-10AVP system equipped with LC10ADVP pump with
SPD-M 10AVP photo diode array detector. The extraction products
obtained were measured on a reversed phase Jupiter C18 column
(250.times.4.6 mm I. D., 5 , 300 .ANG.) (Phenomenex, Part #:
00G-4053-EO, serial No: 2217520-3, Batch No.: 5243-17). The
injection volume was 10 l and the flow rate of mobile phase was 1
ml/min. The column temperature was 50.degree. C. The mobile phase
consisted of A (0.5% aqueous formic acid, v/v) and B
(acetonitrile). The gradient was programmed as follows: with the
first 6 minutes, A maintains at 100%, 6-10 min, solvent B increased
linearly from 0% to 12%, and 10-35 min, B linearly from 12% to 21%,
then 35-40 min, B linearly from 21% to 25%, then 40-50 min, B
linearly to 100%.
[0198] Methanol stock solutions of 3 reference standards (catechin,
epicatechin and Trans-cinnalmaldehyde) were prepared by dissolving
weighted quantities of standard compounds into methanol at 1 mg/ml.
The mixed reference standard solution was then diluted step by step
to yield a series of solutions at final concentrations of 0.75,
0.5, 0.1, 0.05 mg/ml, respectively. All the stock solutions and
working solution were used within 7 days and stored in +4.degree.
C. chiller and brought to room temperature before use. The
solutions were used to identify and quantify the compounds in
cinnamon. Retention times of (+)-catechin (C), (-)-epicatechin
(EC), and trans-cinnamaldehyde (CAN) were about 14.02, 15.22, and
34.00 min, respectively. A linear fit ranging from 0.01 to 10 g was
found. The regression equations and correlation coefficients were
as follows: (+)-catechin: peak area=465303.times.C (g) 5701.4,
R.sup.2=0.9996 (N=6); (-)-epicatechin: peak area=124964.times.C (g)
215.88, R.sup.2=0.9998 (N=6); trans-cinnamaldehyde: peak
area/100=69657.times.C (g)-1162.1, R.sup.2=0.9997 (N=6). HPLC
results are shown in Table 9. The contents of the reference
standards in each sample were calculated by interpolation from the
corresponding calibration curves based on the peak area.
TABLE-US-00009 TABLE 9 HPLC analysis results of cinnamon standard
at concentration of 1 mg/ml in methanol Retention Start Stop time
Area Height Width time time Theoretical ID (min) (mAu min) (mAu)
(min) (min) (min) plate.sup.1 (+)-catechin 14.016 1479356 234337
0.46 13.83 14.29 14854 (-)-epicatechin 15.221 164706 23537 0.64 15
15.64 9050 Trans- 33.984 22590251 1029700 1.66 33.3 34.97 6706
cinnalmaldehyde .sup.1Theoretical plates was calculated by: N = 16
.times. (t.sub.R/w).sup.2. t.sub.R is retention time and w is width
of the peak,
https://www.mn-net.com/web%5CMN-WEB-HPLCkatalog.nsf/WebE/GRUNDLAGEN
GC-MS Analysis
[0199] GC-MS analysis was performed using a Shimadzu GCMS-QP2010
system. The system includes high-performance gas chromatograph,
direct coupled GC/MS interface, electro impact (EI) ion source with
independent temperature control, quadrupole mass filter et al. The
system is controlled with GCMS solution Ver. 2 software for data
acquisition and post run analysis. Separation was carried out on a
Agilent J&W DB-5 fused silica capillary column (30 m.times.0.25
mm i.d., 0.25 m film thickness) (catalog: 1225032, serial No: U.S.
Pat. No. 5,285,774H) using the following temperature program. The
initial temperature was 60.degree. C., held for 2 min, then it
increased to 120.degree. C. at rate of 4.degree. C./min, held for
15 min, then it increased to 240.degree. C. at rate of 4.degree.
C./min, held for 15 min with total running time of 77 minutes. The
sample injection temperature was 250.degree. C. 1 l of the sample
was injected by auto injector at splitless mode in 1 minute. The
carrier gas was helium and flowrate was controlled by pressure at
60 KPa. Under such pressure, the flowrate was 1.03 ml/min and
linear velocity was 37.1 cm/min. MS ion source temperature was
230.degree. C., and GC/MS interface temperature was 250.degree. C.
MS detector was scanned between m/z of 50 and 500 at scan speed of
1000 AMU/second. Solvent cutoff temperature was 3.5 min.
Folin-Ciocalteu Method (Markar 1993) for Total Phenolic Acids
Shimazu UV-V is spectrophotometer (UV 1700 with UV probe: S/N:
A1102421982LP) has been used.
Standard:
[0200] Make stock gallic acid/water solution at concentration of 1
mg/ml. Take suitable amount of gallic acid solution in test tubes,
make up the volume to 0.5 ml with distilled water, add 0.25 ml of
the Folin Ciocalteu reagent and then 1.25 ml of the 20 wt % sodium
carbonate solution. Shake the tube well (untrasonic bath) for 40
min and record absorbance at 725 nm. The data are shown in Table
10. TABLE-US-00010 TABLE 10 Preparations of calibration curve for
gallic acid. Gallic acid Sodium Absorb- solution Gallic Distilled
Folin carbonate ance (0.1 mg/ml) acid water reagent solution at 725
Tube (ml) (.mu.g) (ml) (ml) (ml) mm* Blank 0.00 0 0.50 0.25 1.25
0.000 1 0.02* 2 0.48* 0.25 1.25 0.111 2 0.04 4 0.46 0.25 1.25 0.226
3 0.06 6 0.44 0.25 1.25 0.324 4 0.08 8 0.42 0.25 1.25 0.464 5 0.1
10 0.40 0.25 1.25 0.608 *amount of gallic acid solution is
depending on the absorption information
Direct Analysis in Real Time (DART) Mass Spectrometry for
Polysaccharide Analysis. Instruments: JOEL AccuTOF DART LC time of
flight mass spectrometer (Joel USA, Inc., Peabody, Mass., USA).
This Time of Flight (TOF) mass spectrometer technology does not
require any sample preparation and yields masses with accuracies to
0.00001 mass units. Methods: The instrument settings utilized to
capture and analyze fractions are as follows: For cationic mode,
the DART needle voltage is 3000 V, heating element at 250.degree.
C., Electrode 1 at 100 V, Electrode 2 at 250 V, and helium gas flow
of 7.45 liters/minute (L/min). For the mass spectrometer, orifice 1
is 10 V, ring lens is 5 V, and orifice 2 is 3 V. The peaks voltage
is set to 600 V in order to give resolving power starting at
approximately 60 m/z, yet allowing sufficient resolution at greater
mass ranges. The micro-channel plate detector (MCP) voltage is set
at 2450V. Calibrations are performed each morning prior to sample
introduction using a 0.5 M caffeine solution standard
(Sigma-Aldrich Co., St. Louis, USA). Calibration tolerances are
held to .ltoreq.5 mmu.
[0201] The samples are introduced into the DART helium plasma with
sterile forceps ensuring that a maximum surface area of the sample
is exposed to the helium plasma beam. To introduce the sample into
the beam, a sweeping motion is employed. This motion allows the
sample to be exposed repeatedly on the forward and back stroke for
approximately 0.5 sec/swipe and prevented pyrolysis of the sample.
This motion is repeated until an appreciable Total Ion Current
(TIC) signal is observed at the detector, then the sample is
removed, allowing for baseline/background normalization.
[0202] For anionic mode, the DART and AccuTOF MS are switched to
negative ion mode. The needle voltage is 3000 V, heating element
250.degree. C., Electrode 1 at 100 V, Electrode 2 at 250 V, and
helium gas flow at 7.45 L/min. For the mass spectrometer, orifice 1
is 20 V, ring lens is -13 V, and orifice 2 is 5 V. The peak voltage
is 200 V. The MCP voltage is set at 2450 V. Samples are introduced
in the exact same manner as cationic mode. All data analysis is
conducted using MassCenterMain Suite software provided with the
instrument.
Example 1
Example of Step 1A: Single Step SFE Maximal Extraction and
Purification of Cinnamon Essential Oil
[0203] All SFE extractions were performed on SFT 250 (Supercritical
Fluid Technologies, Inc., Newark, Del., USA) designed for pressures
and temperatures up to 690 bar and 200.degree. C., respectively.
This apparatus allows simple and efficient extractions at
supercritical conditions with flexibility to operate in either
dynamic or static modes. This apparatus consists of mainly three
modules; an oven, a pump and control, and collection module. The
oven has one preheat column and one 100 ml extraction vessel. The
pump module is equipped with a compressed air-driven pump with
constant flow capacity of 300 ml/min. The collection module is a
glass vial of 40 ml, sealed with caps and septa for the recovery of
extracted products. The equipment is provided with micrometer
valves and a flow meter. The extraction vessel pressure and
temperature are monitored and controlled within +3 bar and
-1.degree. C.
[0204] In typical experimental examples, 30 grams of cinnamon bark
powder with size above 105 sieved by 140 mesh screen was loaded
into a 100 ml extraction vessels for each experiment. Glass wool
was placed at the two ends of the column to avoid any possible
carry over of solid material. The oven was preheated to the desired
temperature before the packed vessel was loaded. After the vessel
was connected into the oven, the extraction system was tested for
leakage by pressurizing the system with CO.sub.2 (.about.850 psig),
and purged. The system was closed and pressurized to desired
extraction pressure using the air-driven liquid pump. The system
was then left for equilibrium for .about.3 min. A sampling vial (40
ml) was weighed and connected to the sampling port. The extraction
was started by flowing CO.sub.2 at a rate of .about.10 SLPM (19
g/min), which is controlled by a meter valve. The solvent/feed
ratio, defined as the weight ration of total CO.sub.2 used to the
weight of loaded raw material, was calculated. During the
extraction process, the extracted sample was weighed every 5 min.
Extraction was presumed to be finished when the weight of the
sample did not change more than 5% between two weighing
measurements. The yield was defined to be the weight percentage of
the essential oil extracted with respect to the initial total
weight of the feedstock material loaded into the extraction vessel.
A full factorial extraction design was adopted varying the
temperature from 40-80.degree. C. to 80-500 bar.
[0205] In this experimental example, the extraction conditions were
set wherein the temperatures ranged from 40-80.degree. C. and the
pressures ranged from 80-500 bar. The CO.sub.2 flow rate was 19
g/min. The results are shown in Tables 11. TABLE-US-00011 TABLE 11
HPLC analysis of single stage SFE cinnamon essential oil
extraction. Density CND CND yield T (.degree. C.) P (bar) (g/cc)
S/F Yield (%) purity (%) (%) 40 80 0.293 57 0.46 69.1 0.32 40 100
0.64 57 0.87 60.2 0.53 40 120 0.723 57 0.87 61.5 0.53 40 300 0.915
57 1.27 58.0 0.74 60 80 0.195 38 0.34 65.4 0.22 60 100 0.297 38
0.34 68.1 0.23 60 120 0.448 38 0.43 67.1 0.29 60 300 0.834 38 1.14
58.7 0.67 80 100 0.226 19 0.49 68.0 0.33 80 300 0.751 19 1.14 59.6
0.68
Example 2
Example of Step 1B: Multi-stage SCCO.sub.2 Fractionation of
Cinnamon Essential Oil.
[0206] Multi-stage SCCO.sub.2 extraction/fractionation was
performed using a SFT 250 (Supercritical Fluid Technologies, Inc.,
Newark, Del., USA). In typical multi-stage extractions, 30 g ground
cinnamon bark, particle size greater than 105 m, was loaded into an
extraction vessel with an internal volume of 100 ml. The extraction
solution was collected in a 40 ml collector vessel connected to the
exit of the extraction vessel. The flow rate of CO.sub.2 was set at
19 g/min. The first extraction step was performed at a pressure of
80 bar and a temperature of 40.degree. C. (CO.sub.2 density=0.29
g/ml). This extraction step was carried out for 1 hour. The second
extraction step was performed at a pressure of 100 bar and a
temperature of 40.degree. C. (CO.sub.2 density=0.64 g/ml). The
second extraction step lasted for 1 hour. The third extraction step
was performed at a pressure of 120 bar and a temperature of
40.degree. C. for 1 hour (CO.sub.2 density=0.72 g/ml). A fourth
extraction stage at a temperature of 40.degree. C. and a pressure
of 300 bar (CO.sub.2 density=0.92 g/ml) was then performed for 1
hour. Multi-stage extractions using three stages at 60 C and
80.degree. C. were also performed. The analytical results including
are shown in Table 12 that can be compared with the crude extract
and multi-stage GC-MS data under the same SFE conditions.
TABLE-US-00012 TABLE 12 Multiple stage SFE extraction yield of
cinnamon essential oil. Density Yield stage T (.degree. C.) P (bar)
(g/cc) S/F (%) 1 40 80 0.293 38 0.55 2 40 100 0.64 38 0.55 3 40 120
0.723 38 0.24 4 40 300 0.915 38 0.26 1 60 100 0.297 38 0.60 2 60
300 0.835 38 0.35 3 60 500 0.938 38 0.32 1 80 100 0.227 38 0.75 2
80 300 0.751 38 0.86 3 80 500 0.88 38 0.14
[0207] The total yield of multi-stage extractions at 40, 60, and
80.degree. C. was about 1.6%, 1.3%, and 1.8% by mass weight based
on original feedstock, respectively, by summing up the yield from
each stage. These yields were higher than the yields in the single
stage crude extractions due to a higher solvent-feed ratio that was
used in the multi-stage processing. Otherwise, the data are
consistent. As is apparent from the data, the concentrations of the
chemical constituent chemical compounds such as
trans-cinnamaldehyde can be changed in these sub-fraction
extraction products confirming the ability of SFE to profile the
chemical constituents of cinnamon essential oil. TABLE-US-00013
TABLE 13 Cinnamon essential oil compounds profile in extracts
obtained at different conditions. T = 40.degree. C. T = 60.degree.
C. T = 80.degree. C. Compounds Stage 1 Stage 2 Stage 3 Stage 4
Stage 1 Stage 2 Stage 3 Stage 1 Stage 2 Stage 3 Cinnamaldehyde 67.3
88.0 83.3 67.1 93.1 86.2 74.7 90.7 88.9 74.1 congeners
Sesquiterpenes 1.4 1.5 2.1 2.1 2.7 1.7 2.0 1.1 1.1 3.5 Fatty acids
0.9 2.5 6.6 9.9 0.9 5.9 8.6 1.0 4.1 7.8 and derivatives Steroids
20.3 5.2 0.3 0.8 0.0 0.0 0.0 0.0 0.0 0.0
Example 3
Example of Step 2 Polysaccharide Fraction Extraction
[0208] A typical experimental example of solvent extraction and
precipitation of the water soluble, ethanol insoluble purified
polysaccharide fraction chemical constituents of cinnamon species
is as follows: 20 gm of the solid residue from the SFE extraction
at 60.degree. C. and 300 bar was extracted using 400 ml of
distilled water for two hours at 85.degree. C. in two stages. The
two extraction solutions were combined and the slurry was filtered
using Fisherbrand P4 filter paper (pore size 4-8 m) and centrifuged
at 2,000 rpm for 20 minutes. The supernatant was collected. Rotary
evaporation was used to concentrate the clear supernatant extract
solution from 800 ml to 80 ml. Then, 1520 ml of anhydrous ethanol
was added to make up a final ethanol concentration of 95%. The
solution was allowed to sit for 30 min and a precipitate was
observed. The extraction solution was centrifuged at 2,000 rpm for
20 minutes and the supernatant decanted and either saved for
further processing or discarded. Mass balance was performed before
and after precipitation to calculate the yield of polysaccharides.
The precipitate was collected and dried in an oven at 50.degree. C.
for 12 hours. The dried polysaccharide fraction was weighed and
dissolved in water for analysis of polysaccharide purity with the
colormetric method, using dextran as reference standards. Moreover,
AccuTOF-DART mass spectrometry was used to further characterize the
polysaccharide fraction. The results are shown in FIGS. 6 and 7 and
Tables 14 and 15. TABLE-US-00014 TABLE 14 Precipitated
polysaccharide fraction analysis by water leaching and using 95%
ethanol precipitate. SFE 60.degree. C. and 300 bar residue
Feedstock (g) 20 Water leaching yield (%) 4.8 Leaching extracts
before precipitate (g) 0.96 Leaching extracts after precipitate (g)
0.71 Precipitate (pcp) (g) 0.25 Precipitate yield (%) 1.3 Total
phenolic acid before precipitate (g) 0.25 Total phenolic acid after
precipitate (g) 0.26 Dextran 5K (mg/mg pcp) 0.47 Dextran 50 K
(mg/mg pcp) 0.35 Dextran 410 K (mg/mg pcp) 0.29
[0209] TABLE-US-00015 TABLE 15 DART analysis polysaccharide from
cinnamon. Positive Ion Negative Ion (m + H)/z Relative Intensity (m
- H)/z Relative Intensity 84.28124 99.425442 75.01006 137.56585
86.25373 81.720883 76.98839 5128.816052 93.25277 101.372983
77.12163 118.072363 98.20619 112.664144 87.01636 784.165496
101.1977 179.003571 89.02475 3689.452008 104.2004 74.965155
89.33272 106.713514 110.1915 107.457158 93.0378 98.710896 114.1919
310.219885 94.03036 801.832942 124.1697 541.492879 101.0621
81.171167 127.1837 251.473709 112.0237 132.567353 135.1607
211.982675 113.0289 256.6648 138.1605 184.608718 121.0391
779.921546 143.1455 125.176163 136.0431 1009.934451 146.1552
51.686867 139.0477 321.969773 149.1498 146.588712 151.0508
261.80355 151.1432 124.434696 155.0082 440.154667 152.1561
426.709823 157.0101 177.738929 159.1281 92.057677 165.0284
587.801494 163.1568 508.251143 171.107 197.524616 164.1678
51.884042 176.0796 211.346721 166.156 235.18718 186.0511 116.599949
168.1337 78.968582 187.0408 1166.858983 169.1348 260.595417
188.0499 158.886766 171.1442 59.12023 203.044 112.787336 173.1572
113.644235 205.13 132.109702 176.1467 108.331449 207.1191
131.606635 179.1507 137.84007 215.0732 5416.379733 180.1665
994.055767 215.4763 298.566964 185.1359 150.707896 216.0802
729.308918 186.1501 158.322059 217.0876 99.231184 190.1563
183.096859 221.1137 111.97474 195.175 86.546205 228.0888 100.697547
199.1673 227.035116 230.0733 604.711842 204.1508 71.482813 231.0731
1097.598023 205.156 282.427685 232.0814 111.295636 207.1617
187.039509 234.1212 267.144226 209.1427 76.891885 235.1485
202.94284 212.1917 121.104614 247.0839 111.958677 217.1726
778.327585 347.5377 55.470255 218.1681 219.204541 353.1065
49.397762 222.1693 68.666762 374.1498 462.267476 223.091 736.949569
381.5363 65.488965 225.161 83.791408 227.1636 179.282801 234.1969
351.374295 235.1968 221.299761 237.171 165.239214 244.1914
173.437145 253.1741 170.977467 255.2016 151.941156 257.2369
211.908424 269.2121 633.77052 270.2101 154.111628 271.2321
1124.577818 272.2465 339.732994 273.2465 1044.173233 274.2533
215.595509 279.1588 1902.133282 280.1583 320.860255 281.2123
96.009582 283.2191 1281.201573 284.2204 240.818719 285.2101
533.098708 286.2286 253.564416 287.2236 1550.802257 288.2426
3224.93612 289.2434 3384.734363 290.2552 810.746561 291.2548
287.438135 293.2133 105.940041 295.2189 297.089805 297.2621
150.897354 298.2583 58.674498 299.2333 353.940052 300.28 277.224355
301.2168 662.198609 302.2438 316.351463 303.2279 1157.364552
304.2443 2292.402403 305.2408 3391.780079 305.5312 171.674883
306.2432 871.724411 307.2501 5097.759878 307.5592 135.272649
307.8653 67.055551 308.2576 1307.87184 309.2461 276.320258 314.2569
196.483658 315.2256 218.14155 316.2783 914.795178 317.2599
331.764991 318.2375 59.32597 319.2205 718.902252 320.2407 260.17705
321.235 2454.356967 322.2501 822.288344 323.2512 2417.876001
324.264 599.186884 325.2689 204.666646 331.2688 147.777759 335.2215
345.293408 336.2407 147.720225 337.2279 1077.500668 338.2533
412.261973 339.2448 1476.416047 340.2592 514.704806 344.3092
193.613385 345.227 60.106943 347.2457 2194.894335 348.2636
711.622206 349.2606 4190.740285 350.262 988.545178 351.2579
404.799383 353.2215 300.675765 354.2486 152.247089 355.2466
416.642895 356.259 552.671805 357.2717 201.754991 361.2327
90.263863 363.2422 1061.838748 364.2584 266.66125 365.2561
1352.426638
[0210] The cinnamon polysaccharide yield was 1.3% by mass weight
based on the original cinnamon bark feedstock. The purity of the
polysaccharide fraction was 290-470 mg/g dextran standard
equivalent indicating a purity of >95% cinnamon polysaccharide
chemical constituents in the fraction. Comparing the analysis of
total phenolic acids in solution before and after the
precipitation, the precipitation appeared to have no effect on the
phenolic acids. Based on a large number and variety of experimental
approaches, it is quite reasonable to conclude that 1.3% yield is
almost 100% of the water soluble-ethanol insoluble polysaccharides
in the natural cinnamon species feedstock material.
Example 4
Example of Step 3: Hydroalcoholic Leaching Extraction
[0211] A typical example of a 3 stage solvent extraction of the
phenolic acid chemical constituents of cinnamon species is as
follows: The feedstock was 2 gm of ground cinnamon bark SFE residue
from Step 1 SCCO.sub.2 (40.degree. C., 300 bar) extraction of the
essential oil. The solvent was 40 ml of 25% aqueous ethanol. In
this method, the feedstock material and 40 ml aqueous ethanol were
separately loaded into 100 ml extraction vessel and mixed in a
heated water bath at 40.degree. C. for 4 hours. The extraction
solution was filtered using Fisherbrand P4 filter paper having a
particle retention size of 4-8 m, centrifuged at 2000 rpm for 20
minutes, and the particulate residue used for further extraction.
The filtrate (supernatant) was collected for yield calculation and
HPLC analysis. The residue of Stage 1 was extracted for 2 hours
(Stage 2) and the residue from Stage 2 was extracted for 2 hours
using the aforementioned methods. The supernatants were collected
for mass balance, HPLC analysis for cinnamaldehyde (CND), catechin
(C), and epicatechin (EC) in the extracts. Folin-Ciocalteu assay
was used for measuring total phenolic acid concentration (purity)
and protein precipitation method was used for measuring tannin acid
purity. The results are shown in Table 16. TABLE-US-00016 TABLE 16
Effect of multiple hydroalcoholic leaching stages on extraction
yield Purity (%) Yield (%) Stage Yield (%) CND C EC TPA TA CND C EC
TPA 1 11.05 4.66 2.33 15.75 63.26 14.8 0.52 0.26 1.74 6.99 2 6.56
8.20 3.00 18.42 65.39 23.1 0.54 0.20 1.21 4.29 3 0.41 5.73 2.98
16.51 51.44 81.7 0.02 0.01 0.07 0.21 Note: 1. CND =
trans-cinnalmaldehyde; C = (+)-catechin; EC = (-)-epicatechin; TPA
= total phenolic acid; TA = tannin acid. 2. CND, C, EC were
analyzed by HPLC; TPA was analyzed by Folin-Ciocalteu method by
using Gallic acid as standard; TA was analyzed by
protein-precipitation method.
[0212] In order to verify Folin-Ciocalteu method, known phenolics
acid, kaempherol, caffeic acid, catechin, at concentration of 1
mg/ml were tested. The experimental error measuring kaempherol and
catechin was in the order of 2-4% and that in caffeic acid case was
about 10%. In addition, one reference (Sindhu 2006) tested total
phenol acid in their method extracts and the results was
289.sub.--2.2 mg gallic acid/g extracts, which is fairly close to
the present results.
Example 5
Example of Step 4 Affinity Adsorbent Extraction of Purified
Polyphenolic Acid Fraction
[0213] In typical experiments, the working solution was the
transparent hydroalcoholic solution of cinnamon species aqueous
ethanol leaching extract in Step 3. The affinity adsorbent polymer
resin was Sephadex LH-20. 6 gm of affinity adsorbent was pre-washed
with 95% ethanol (4-5 BV) before packing into a column with an ID
of 1.5 cm and length of 100 cm. The packed column volume was 25 ml.
100 ml of cinnamon 25% ethanol stage I+stage II extraction solution
(sample solution. 2.4 mg/ml) was concentrated to 1 ml using rotary
evaporation to remove the solvent. Then, 19 ml of absolute ethanol
was added to the concentrated solution to dissolve the chemical
constituents. This solution was centrifuged at 2000 rpm for 10
minutes and the supernatant collected as the final polyphenolic
loading solution (11 mg/ml). 12 ml of the loading solution was
loaded onto the column. The loaded column was eluted with 240 ml of
95% ethanol at a flow rate of 2.4 BV/hr (1 ml/min) with an elution
time of 100 minutes. During elution, 8 non-tannin polyphenolic
fractions were collected (labeled Elution Fraction F1-F8) at each
30 ml of elution. Each fraction was tested using UV
spectrophotometry at 280 nm until the absorbance could no longer be
detected in the fraction collected. The column washed with 70 ml of
70% aqueous acetone to remove the tannin polyphenolics adsorbed on
the affinity adsorbent at a flow rate of 5 BV/hr (2.1 ml/min). The
tannin washing solution was discarded. Finally, the column washed
with 4-5 BV of 95% ethanol to remove any remaining chemical
impurities in order to prepare the column for further processing.
Each polyphenolic elution fraction was collected and analyzed and
the results are shown in Table 17. TABLE-US-00017 TABLE 17 Analysis
of 95% ethanol elutions of polyphenolic fractions from Sephadex
LH-20 process chromatography. Weight (mg) Purity (%) Total Non
Yield Total phenolic Tannin Nontannin Tannin tannin Average Name
(%) solid acid acid acid acid acid DPn Loading 132.1 61.2 32.8 28.5
21.6 24.8 6.9 Elution F2 37.1 49.0 3.7 0.1 3.6 7.1 0.1 3.6 Elution
F3 7.4 9.8 2.9 0.0 2.9 29.5 0.0 2.7 Elution F4 5.2 6.8 4.5 0.0 4.5
66.4 0.0 3.6 Elution F5 3.2 4.2 3.7 0.0 3.7 87.8 0.0 3.1 Elution F6
2.3 3.1 2.9 0.0 2.9 91.1 0.0 4.0 Elution F7 2.1 2.8 2.9 0.0 2.9
100.0 0.0 4.2 Elution F8 1.2 1.6 1.6 0.0 1.6 93.8 0.0 4.2 Combine
5.7 7.5 7.3 0.0 7.3 97.2 0.0 4.1 + 0.1 F6-F8 Combine 21.5 28.4 18.5
0.0 18.5 65.1 0.0 3.6 .perp. 0.6 F2-F8 Recovery 58.5 36.1 0.2 77.4
(%) * Elution 1 was not tabulated because there were no chemical
constituents, only solvent.
Example 6
[0214] The following ingredients are mixed for the formulation:
TABLE-US-00018 Extract of C. cassia bark 150.0 mg Essential Oil
Fraction (10 mg, 6.6% dry weight) Polyphenolic Fraction (100 mg,
66.7% dry weight) Polysaccharides (40 mg, 26.6% dry weight)
Stevioside (Extract of Stevia) 12.5 mg Carboxymethylcellulose 35.5
mg Lactose 77.0 mg Total 275.0 mg
[0215] The novel extract of cinnamon species comprises an essential
oil fraction, phenolic acid-essential oil fraction, and
polysaccharide fraction by % mass weight greater than that found in
the natural rhizome material or convention extraction products. The
formulations can be made into any oral dosage form and administered
daily or to 15 times per day as needed for the physiological and
psychological effects desired (enhanced brain function and
analgesia) and medical effects (non-insulin dependent diabetes
mellitus, anti-platelet aggregation and anti-thrombosis,
cardiovascular and cerebrovascular disease prevention and
treatment, anti-atherosclerosis, anti-hypercholesterolemia, cardiac
protection, nervous system protection, anti-inflammatory,
anti-allergic, anti-arthritis, anti-rheumatic, anti-gout,
gastro-intestinal disorders, cough, common cold, fever, lipolytic,
improved wound healing, anti-bacterial, anti-fungal, and
anti-cancer).
Example 7
[0216] The following ingredients were mixed for the following
formulation: TABLE-US-00019 Extract of C. cassia 150.0 mg Essential
Oil Fraction (60 mg, 40% dry weight) Polyphenolic Fraction (30 mg,
20% dry weight) Polysaccharides (60.0 mg, 40% dry weight) Vitamin C
15.0 mg Sucralose 35.0 mg Mung Bean Powder 10:1 50.0 mg Mocha
Flavor 40.0 mg Chocolate Flavor 10.0 mg Total 300.0 mg
[0217] The novel extract of cinnamon chuangxiong comprises an
essential oil, phenolic acid-essential oil, and polysaccharide
chemical constituent fractions by % mass weight greater than that
found in the natural plant material or conventional extraction
products. The formulation can be made into any oral dosage form and
administered safely up to 15 times per day as needed for the
physiological, psychological and medical effects desired (see
Example 1, above).
REFERENCES
[0218] 1. Khan A et al. Diabetes Care 26:3215-3218, 2003. [0219] 2.
Anderson R A et al. J Agric Food Chem 52:65-70, 2004. [0220] 3.
Jarville-Taylor et al. J Am Coll Nutri 20:327-336, 2001. [0221] 4.
Qin R et al. Horm Metab Res 36:119-123, 2004. [0222] 5. Vespohl E J
et al. Phytother Res 19:203-206, 2005. [0223] 6. Lee S H et al
Biochem Pharmacol 69:791-9, 2005. [0224] 7. Chericoni S et al. J
Agric Food Chem 53:4762-4765, 2005. [0225] 8. Lin C C et al.
Phytother Res 17: 7260730,2003. [0226] 9. Jayaprakasha G K et al. J
Agric Food Chem 51:4344-4348, 2003. [0227] 10. Huss U et al. J Nat
Prod 65:1517-21, 2002. [0228] 11. Nagai H et al. Jpn J Pharmacol
32:813-822, 1982. [0229] 12. Su M J et al. J Biomed Sci 6:376-386,
1999. [0230] 13. Shimada Y et al. Phytomed 11:404-410, 2004. [0231]
14. Taher M et al. Med J Malayia 59B:97-98, 2004. [0232] 15. Kamath
J V et al. Phytother Res 17:970-972, 2003. [0233] 16. Kurokawa M et
al. Eur J Pharmacol 348:45-51, 1998. [0234] 17. Simic A et al.
Phytother Res 18:713-717, 2004. [0235] 18. Tabak M et al. J
Ethnopharmacol 67:269-277, 1999. [0236] 19. Kong L D et al. J
Ethnopharmacol 73:199-207, 2000. [0237] 20. Kwon B M et al. Arch
Pharm Res 21:147-152, 1998. [0238] 21. Ka H et al. Cancer Lett
196:143-152, 2003. [0239] 22. Williamson E M. Phtomedicine
8:401-409, 2001. [0240] 23. Dubois M et al. Analytical Chem
28:350-356, 1956. [0241] 24. Gu L et al. J Agric Food Chem
51:7513-7521, 2003. [0242] 25. Guyot S et al. Methods in Enzymology
335:57-70, 2001. [0243] 26. Maria Jerez P et al. Food Chem
94:406-414, 2006. [0244] 27. Makkar H P S et al. J Sci Food Agric
61:161-165, 1993. [0245] 28. Makkar H P S et al. J Agric Food Chem
36:523-525, 1988.] [0246] 29. Shindu, M. Food Chem 94:520-528,
2006.
* * * * *
References